1367S

ENHANCED PRODUCTION OF ETHANOL FROM
SUGAR CANE MOLASSES THROUGH THERMOTOLERANT

SACCHAROMOMYCESCEREVISIAE CELL
A thesis submitted by
SYED FARMAN ALI SHAH
In fulfillment of the requirement for the degree of

Doctor of Philosophy
in
Chemical Engineering
Department of Chemical Engineering

Faculty of Engineering
Mehran University of Engineering and Technology
Jamshoro
June 2010
DEDICATION
To Holy Prophet
Hazrat Muhammad Mustafa
Salallahu Alaihi Wa Alihi Wa Sallam
ii
iii
ACKNOWLEDGEMENTS
Allah, The omnipotent, The beneficent and The merciful, Who created the universe
and bestowed mankind with the knowledge and ability to think into His secrets.
Peace and blessings of Allah be upon Holy Prophet Muhammad (salallahu alaihi wa
aalihi wasallam, the unique comprehensive personality, the everlasting source of
guidance and knowledge for humanity, I thank to all.
I would like to thank my supervisor, Late Professor Dr. Muhammad Ibrahim Pathan,
Former Dean Faculty of Engineering, Mehran University of engineering and Technology
(MUET), Jamshoro. He rendered his valued services to the noble cause of education. I
pray for his departed soul.
Gratitudes for Professor Dr. Hafeez-ur-Rahman Memon,Director,Institute of Petroleum
and Natural Gas Engineering, MUET, Jamshoro, for his kind supervision after the
death of Dr.Pathan.
Thanks to Professor Dr.Abdul Qadeer Khan Rajput,The Vice Chancellor, Professor Dr.
Abdul Ghani Pathan, Dean Faculty of Engineering, Professor Dr. Ghous Bakhsh
Khaskheli, Director and Mr. Mahboob Ali Abbasi, Assistant Registrar, Postgraduate
Studies.
I am deeply indebted to my Honorable co-supervisor Dr Muhammad Ibrahim Rajoka,
Deputy
Chief
Scientist,
National
Institute
iv
for
Biotechnology
and
Genetic
Engineering (NIBGE) Jhang Road Faisalabad for the continuous help, support and
stimulating suggestions and encouragement during my Ph.D. research work in NIBGE
Faisalabad. Thanks to Mr. Muhammad Ferhan, Junior Scientist, NIBGE, Faisalabad am
also thankful to Mr.Ali Ahmed and Ms. Munazza Afzal, who imparted the heights of
their assistance in my laboratory work, which helped me a lot.
I feel a deep sense of gratitude for my family, who felt difficulties in my absence from
home during my research work but always prayed for my success.
Lastly, I am grateful to my family and friends for the inspiration and moral support they
provided during my Ph.D. work.
TABLE OF CONTENTS
Description
Page#
List of Notations
xii
List of Abbreviations
xiv
List of Tables
xvii
List of Figures
xxi
Abstract
xxviii
CHAPTER 1 INTRODUCTION
1.1 General
1.2 Ethanol and its scope
1.3 Ethanol production
1.4 Raw materials for ethanol production
1.5 Fermentation by yeast, S. cerevisiae
1.6 Aeration in fermentation
1.7 Oxygen transfer
1.8 Determination of KLa value
vi
1.9 Why thermotolerant yeast is used?
1.10 General modification of strain
1.11 Objectives of present research
10
CHAPTER 2 LITERATURE REVIEW
12
2.1 Ethanol and its by-products
12
2.2 Yeast and invertases
18
2.3 Other parameters
20
2.3.1 Substrate concentration
20
2.3.2 Nitrogen and carbon sources
20
2.3.3 Airflow rate
20
2.3.4 Additives
20
2.3.5. Thermodynamics of ethanol and Ffase
21
formation
CHAPTER 3 MATERIALS AND METHODS
26
3.1 Research centers
26
3.2 Sample centers
26
3.3 Microbial strain
26
3.4 Maintenance of culture
27
vii
3.5 Growth medium composition
28
3.6 Preparation of plates
28
3.7 Preparation of slants
29
3.8 Preparation of the culture of native S. cerevisiae
30
3.8.1.
Preparation of yeast growth medium
30
3.9 Effect of gamma rays irradiation on viability of
32
cells
3.10 Selection of mutant of S. cerevisiae
32
3.11 Purification of mutants of S. cerevisiae
33
3.11.1.
Slants of purified culture
3.12 Propagation of yeast
33
34
3.12.1
First stage propagation
34
3.12.2
Second stage propagation
38
3.12.3
Fermentation
38
3.13 Effect of carbon and nitrogen sources on ethanol
39
production
3.14 Effect of different additives on ethanol yield by
39
both wild and mutant culture in 23 liter fermenter

3.15 Effectiveness of air in ethanol production
39
3.16 Analytical methods
40
viii
3.16.1
Preparation of standard curve for
40
biomass estimation
3.16.2
Biomass estimation
41
3.16.3
Extraction of ethanol
41
3.16.4
Ethanol estimation through
42
HPLC
3.16.5
Harvesting of intracellular
43
invertases
3.16.5.1
Invertase assay
43
3.16.5.2
Determination of units for
44
invertase activity
3.16.6
Glucose concentration
45
determination
3.16.6.1
Preparation of DNS
45
(Dinitrosalicylic Acid) solution

3.16.6.2
3.16.7
Standard curve of glucose
46
Substrate utilization
46
3.17 Chemical composition of hydrol (starch molasses).
47
3.18 Determination of growth kinetic parameters
49
3.19 Effect of temperature
50
3.20 Determination of thermodynamic parameters
51
ix
3.20.1 Thermodynamics of cell mass and
51
product formation
3.20.2 Thermodynamics of ethanol formation
3.21. Effect of pH
51
52
CHAPTER 4 RESULTS AND DISCUSSION
53
4.1 Mutagenesis of S. cerevisiae using -rays
53
4.2 Substrate regulation of invertase and ethanol
54
production
4.3 Initial observations
64
4.4 Substrate concentration dependent formation of
66
ethanol
4.5 Effect of substrate sources
75
4.6 Regulation of ethanol production by nitrogen
79
sources
4.7 Effectiveness of air on productivity of ethanol
85
4.8 Effect of Agitation
90
4.9 Effect of additives
95
4.10 Production from hydrol

4.10.1.
99
Effect of temperature on ethanol

production from molasses.
108
4.11 Thermodynamics of ethanol production process

4.11.1
Thermodynamics of ethanol production
114
114
from hydrol and molasses

4.11.2.
Thermodynamic parameters of extra-
119
cellular Ffase production

4.12 Effectiveness of pH for alcohol & Ffase
122
production
4.13 Ethanol and Ffase in 150 liter fermenter for the
123
productivity
CHAPTER 5 CONCLUSIONS & RECOMMENDATIONS
129
5.1
CONCLUSIONS
129
5.2
RECOMMENDATIONS
132
134
REFERENCES
xi
LIST OF NOTATIONS
Liquid phase mass transfer coefficient
Total surface area available for mass transfer
Concentration driving force
Feed rate for substance
kDa
Kilo Dalton
td
doubling time
nm
nanometer
Specific growth rate (slope of ln x v/s time)
Qx
g cells per litre per hour
Qs
g substrate consumed per litre per hour
Qp
g product per hour per litre
Yx / s =
g cells per gram substrate consumed
Yp/x
g ethanol per gram cell
Yp/s
g ethanol per gram substrate consumed
Qp
g ethanol per gram cells per hour
Qs
Specific ethanol yield in g ethanol per g substrate per hour
Cells (g per litre)
Product (ethanol g/l)
xii
Substrate
TY
Yield % (Based on maximum yield of ethanol per substrate (0.51 g

Ethanol per g glucose)
xiii
LIST OF ABBREVIATIONS
CFU
Colony Formation Unit
CSL
Corn Steep Liquor
Dilution rate
DAP
Di Ammonium Phosphate
DNA
Deoxy Nitrocelicellic Acid
DNS
Di Nitro Sulphate
DO
Dissolved Oxygen
E85
Blend of 85% ethanol and 15 % gasoline
EtOH
Ethanol
FPL
Fast Product Laboratory
GRAS
Generally Regarded as Safe
hour
HCl
Hydrochloric Acid
HPLC
High Performance Liquid Chromatography
Hsp
Heat Shock Proteins
HSuc
High Concentration Sucrose
ICR
Immobilized Cell Reactor
IU
International Units
xiv
Liter
Mass
MSW
Municipal Solid Waste
NaCl
Sodium Chloride
NaF
Sodium Fluoride
NaOH
Sodium Hydroxide
NIBGE
National Institute for Biotechnology and Genetic Engineering
OD
Optical Density
OTR
Oxygen Transfer Rate
OUR
Oxygen Uptake Rate
PAGE
Poly Acryl amide Gel Electrophoresis
PAEC
Pakistan Atomic Energy Commission
ppm
Parts per million
PSI
Proteomic Standard Initiative
RPM
Revolutions per Minute
RQ
Respiratory Quotient
Substrate or Glucose content
SOUR
Specific Oxygen Uptake Rate
Time
TRS
Total Reducing Sugars
xv
UV
Ultra Violet
Volume
VF
Final Volume
Vi
Initial Volume
xvi
LIST OF TABLES
DESCRIPTION
PAGE #
Table 1.1
Important physical properties of ethanol
Table 1.2
Major substrates for fermentative production of ethanol
Table 3.1
Chemical composition of growth medium:
28
Table 3.2
Chemical composition of inoculum medium
30
Table 3.3
Concentration of glucose for standard curve
46
Table 3.4
Physico-chemical characteristics of hydrol (starch molasses)
48
Table 4.1
Comparative fermentation kinetic parameters of S. cerevisiae
62
and its mutant derivative M9 for extra-cellular and

intracellular Ffase production and specific growth rate
on different concentrations of sucrose in 23l fermentor
(working volume 15l) at 30 C
Table 4.2
Comparative fermentation parameters of S. cerevisiae and its
63
mutant derivative M9 for production of extracellular and

intracellular Ffase production on different substrates in 23 l
fermenter at 30 C
Table 4.3
Kinetics parameters for cell mass production substrate

consumption and ethanol formation by the native (N) and
mutant (M) cells of S. Cerevisiae at different concentrations
of sugars.
xvii
70
Table 4.4
Time dependent molasses concentration on ethanol and cell
72
mass formation
Table 4.5
Substrate concentration dependent kinetics parameters for
72
ethanol and cell mass formation and substrate utilization by

the mutant organism calculated using data in Table 4.4.
Table 4.6
Effect of different substrate sources on ethanol and cell
77
mass formation with time dependent substrate

consumption from both sources
Table 4.7
Kinetics for mutant strain for substrate consumption and
78
product formation parameters from molasses and hydrol

Table 4.8
Time dependent effect of different nitrogen sources on
81
ethanol, cell mass and substrate present in 15 liter

working volume fermenter.
Table 4.9
Kinetics of product formation of ethanol and substrate
82
consumption parameters for mutant strain of S. cerevisiae

using N-sources.
Table 4.10
Effectiveness of air flow on ethanol and cell mass production
85
during time course uptake of sugars from molasses

Table 4.11
Airflow rate dependent kinetic parameters for ethanol

formation and substrate consumption by the mutant strain by
maintaining all other process variables constant
airflow rate, which had different values.
xviii
except
86
Table 4.12
Effectiveness of agitation for ethanol productivity and for the
91
growth of cells during the time dependent substrate uptake

standard working conditions
Table 4.13
Agitational dependent kinetics parameters of mutant strain
92
for substrate consumption, and product formation in 15 liter

(working volume) fermenter under standard working
conditions.
Table 4.14
Effect of different additives on ethanol and cell mass
96
formation with time dependent consumption of 15 %

total sugars in molasses (pH =5.5) under optimized
working conditions
Table 4.15
Kinetic parameters of
S. cerevisiae M -9 for ethanol
103
production following growth on 15 % sugars in Dextrozymepretreated hydrol in a fully controlled 23-l fermenter
Table 4.16
Temperature effect on biomass formation and substrate
104
Consumption by S. cerevisiae M-9 during ethanol

production following growth on 15 % sugars in Dextrozymepretreated hydrol in a fully controlled 23-l fermenter
Table 4.17
Effect of different representative temperatures on ethanol

and cell mass formation during time-dependent sugar
consumption of sugars from molasses medium by both wild
and mutant strains of S. cerevisiae
xix
110
Table 4.18
Temperature-dependent kinetic parameters for ethanol and

cell mass formation during substrate consumption
111
(15%
total sugars in molasses, pH=5.5) by the mutant strain of

S. cerevisiae
Table 4.19
Enthalpy and entropy values of ethanol production and
118
inactivation pathways following growth on hydrol and

molasses
Table 4.20
Enthalpy and entropy values of extracellular Ffase

production and inactivation
122
pathway following growth
on molasses at different temperatures

Table 4.21
Dependence of ethanol production on culturing condition

namely 23 liter, 150 liter fermenter and shake flask (s. flask)
cultures: Kinetic parameters for substrate consumption
(molasses) and ethanol formation by S. cerevisiae mutant
derivative M- 9.
xx
127
LIST OF FIGURES
DESCRIPTION
PAGE#
Fig. 3.1
Plate culture of fresh thermotolerant S.cerevisiea
27
Fig.3.2
A view of the 23 Liter Fermenter
36
Fig3.3
A view of a 150 Liter Fermenter
37
Fig.3.4
HPLC chromatograms of fermented molasses and untreated
42
hydrol containing 15 % (TS)

Fig.3.4
HPLC chromatogram of properly diluted and filtered
48
hydrol using RI detector.

Fig.4.1
Protein expression profile with and without sucrose in the
58
growth medium for both wild and derepressed mutant stains

of Sachharomyces cerevisiae.
Fig. 4.2(a)
Extracellular -fructo-furanosidase (Ffase) by parental cells
59
(), and mutant cells () and intracellular FFase by parental

() and mutant cells () following growth on 8 % sucrose in
yeast medium which carries substrate.
Fig.4.2(b)
Extracellular -fructo-furanosidase (Ffase) by parental cells

(), and mutant cells () and intracellular FFase by parental
() and mutant cells () following growth on 10 % sucrose
xxi
60
in yeast medium which carries substrate.

Fig.4.3
Kinetics of production of ethanol (), cell mass () and
61
substrate in the medium (), extracellular (inverted open

triangle) and intracellular () following growth of mutant
cells body.
Fig.4.4(a)
Effect of sugar concentrations [ 5 %( , ), 8 %( , ) 10

%(,), 12 %(,) and 15 %(
67
, )] in molasses on
ethanol production by native (empty symbols), and mutant

(filled thick symbols) cells of S. cerevisiae
Fig.4.4(b)
Effect of sugar concentrations [ 5 %(, ), 8 %(, ) 10
68
%(,), 12 %(,) and 15 %( ,)] in molasses on

cell mass production by native (empty symbols), and
mutant (filled thick symbols) cells of S. cerevisiae
Fig.4.4(c )
Effect of sugar concentrations [ 5 %(,), 8 %( , ) 10

%(,), 12 %(,) and 15 %(
69
,)] in molasses on
substrate consumption by native empty symbols), and

mutant (filled thick symbols)) cells of S. cerevisiae
Fig.4.5(a)
Effect of sugar concentration on the production of

ethanol by the wild (open symbols) and mutant
derivative of S. cerevisiae in 15 liter working volume
xxii
73
fermenter. Each value is a mean of two observations

Fig.4.5(b)
Effect of sugar concentration on cell mass formation by the
74
native (open symbols and mutant derivative of the test

organisms
Fig.4.5(c )
Effect of sugar concentration on substrate consumption by
75
both native (open symbols) and mutant derivative (closed

symbols) done as described in materials and methods.
Fig.4.6(a)
Effect of substrate sources on cell mass formation by the
78
native
Fig.4.6(b)
Effect of substrate sources on substrate consumption by both

organisms, wild and mutated.
79
Fig.4.7(a)
Ethanol production from 30 C by both wild (open symbols)
82
and mutant (closed symbols) strains in 23 liter working

volume fermenter. All conditions were kept constant except
nitrogen sources were altered and maintained at a
concentration of 0.246 % nitrogen.
Fig.4.7(b)
83

concentration of 0.246 % nitrogen
Fig.4.7(c )

xxiii
84

concentration of 0.246 % nitrogen
Fig.4.8(a)
Ethanol by both Native and mutated strains of S. cerevisiae at
87
30 C All other process variables were kept constant except

air flow rate, which was changed from 0.1 to 0.4 vvm.
Fig.4.8(b)
Cell growth by both Native and mutated strains of S.
88
cerevisiae at 30 C all other process variables were kept

constant except air flow rate, which was changed from 0.1 to
0.4 vvm.
Fig.4.8( c)
Substrate Consumption by both Native and mutated strains
89
of S. cerevisiae at 30 C All other process variables were

kept constant except air flow rate, which was changed from
0.1 to 0.4 vvm.
Fig.4.9(a)
Effectiveness of agitation for ethanol productivity from 15 %
93
total sugars from both of the organism. The data given in the
table is an average of the two readings
Fig.4.9 (b)
Effect of agitation (rpm) on cell mass formation from 15%
94
total sugars The data given in the table is an average of the

tow readings .The errors between values were small there
fore it is not shown in the data.
Fig.4.9(c )
Effect of agitation on substrate consumption from 15% total

sugars in molasses from the organism. The data given in the
table is an average of the tow readings .The errors between
xxiv
95
values were small there fore it is not shown in the da

Fig.4.10(a)
Effectiveness of additives shown for ethanol productivity

from molasses (total sugars 15 %, pH =5.5) under optimized
working conditions.
97
Figure 4.10b
Effect of additives on cell mass synthesis from molasses

(total sugars 15 %, pH =5.5) under optimized working
conditions. Additives were Tween 80 for parental () and
mutated () strain respectively.
98
Fig.4.10(c )
Effect of additives on substrate consumption by the native
99
(open symbols) and mutant strain (closed symbols) of S.

cerevisiae.
Fig.4.11
Effect of substrate concentration on specific growth rate
100
(), specific substrate consumption (qS), volumetric rate of

product formation (QP), product yield (YP/S) and
specific productivity (qP) in 23-l fermenter using
Dextrozyme-pretreated hydrol as substrate, and corn steep
liquor (25 g/l) as nitrogen source. Initial flow rate was
1 vvm for 8 h followed by 0.25 vvm in agitated vessel
(250 rpm) at 30 C.
Fig.4.12
Determination of activation energy for growth (a) and
105
formation of Ethanol with the help of native and the mutated

strain. using hydrol based medium (dextrozyme treated 15%
sugars containing hydrol) using Arrhenius relationship.
Fig.4.13
Intracellular protein expression profile of derepressed and
107
thermotolerant mutant M-9 on 15% TS with 3% corn steep

liquor (lanes 1-2), and native culture on this medium (lanes
3-4). M= protein marker and invert = standard invertase
from Sigma-Aldrich
Fig.4.14(a)
Effect of temperature on the production of ethanol from
xxv
112
under optimized conditions

Fig.4.14(b)
Effect of temperature on cell mass formation from 15%

sugars
113
Fig.4.14(c)
Effect of temperature on substrate consumption from 15%

sugars
114
Fig.4.15(a)
Enthalpy and entropy requirements for alcohol production
116
from hydrol in the shown temperature ranges. An average is

shown in the data, = parental and = mutant culture as per
Arrhenius equation.
Fig. 4.15(b)
Enthalpy and entropy requirements for alcohol production
117
from hydrol in the shown temperature ranges. An average is

shown in the data, = parental and = mutant culture as per
Arrhenius equation.
Fig. 4.16
Arrhenius relationship to calculate enthalpy and entropy of

activation
for
invertase
production
and
121
inactivation
pathway
Fig.4.17
Effect of controlled pH on Ffase formation by parental ()
124
and mutant () culture and ethanol production by the

parental () and mutant () cultures respectively from
15% sugars in molasses at 30 C under optimized
conditions of aeration and agitation.
Fig.4.18
Representative time course production of ethanol by native

() and mutant (), cell mass by native () and
xxvi
125
mutant () with consumption of sugars by native

() and mutant ()in 150 liter fermenter using
hydrol as a carbon source at 40 C.
xxvii
ABSTRACT
In the present study, Sachharomyces cerevisiae produced invertase and ethanol from
different C-sources and TS. It was catabolite repression sensitive but could grow up to 40
C, though maximum growth and product formation occurred at 30-35 C. The -rays
mutagenesis of Sachharomyces cerevisiae was carried out at 1.2 kGy to select catabolite
repression resistant mutant derivative with retention of its ability to hyperproduce ethanol
and invertase at 43 C. Production of ethanol and invertase by Sachharomyces cerevisiae
wild and its 2-deoxy-D-glucose (DG) resistant mutant (M9) was optimized involving oneat-a-time approach. The mutant M9 also hyper-produced both ethanol and invertase from
sucrose and molasses-based media. A concentration of 15 % total sugars in molasses was
optimized as the best sugar concentration which produced 74 g/l ethanol in 23 litre
fermenter (working volume 15 litre). Lower concentrations resulted in lower values and
higher sugar concentrations needed more time for complete fermentation. Because of
better results on molasses medium with 15 % total sugars (TS), it was adopted regarding
these studies
CSL was used as N-source and as the only supplement and produced 75 g/l ethanol; 9.4
g/l cell mass and consumed 148 g/l of the sugars. The addition of NaF and Tween 80 as
additives did not show any encouraging results, however, Tween 80 proved better if
utilized for more time up to 72 h.
xxviii
Studies have a firmed observation that more than 96% TS are utilized at a rpm of 250300 and an optimized rate of oxygen for the maximizing ethanol production.
This mutant of Saccharomyces cerevisiae was employed for ethanol production from
starch-based concentrate (locally called hydrol), in 23-l fermenter for optimization of
process variables by optimizing one variable at a time approach. Maximum ethanol was
attained at 36 h of cultivation of dextrozyme-treated hydrol under optimized fermentation
conditions (sugars 150 g/l; Dextrozyme 1.0 unit/g maltose, maltotriose and
polysaccharides; pH 5.5; ammonium sulphate 10 g/l and temperature 40 C). The
maximum rates i.e., (YP/S) were 2.82 g/g cells h and 0.49 g/g respectively. Determination
of activation energy for cell growth (Eag= 20.8 kJ/mol) and death (Egd= 9.1 kJ/mol) and
product formation and inactivation (EP=35.8 kJ/mol and Edp=33.5 kJ/mol) revealed the
thermo-stability of the organism up to 47 C and can be exploited in a wide temperature
range (in summer) for ethanol production.
Thermodynamic studies revealed that mutation had thermostablization influence on the
growth, ethanol and enzymes production equilibria. The mutant M9 required lower
activation energy (Ea(P)) Gibbs free energy (G*P), enthalpy (H*P) and entropy (S*P)
magnitudes for ethanol and invertase formation. The activation enthalpy of ethanol and
Ffase formation equilibria by the mutant was lesser in values for ethanol and Ffase
production. In activation pathway were quite comparable and are the criteria of
thermostable metabolic network of thermophilic organisms. The mutant strain is better in
xxix
the inactivation equilibria. Mutation made the organism significantly better with respect
to genetic make up in the glycolytic pathway of the organism.
When molasses and Enzoz hydrol were compared, molasses proved better (74 g/l of
ethanol) than Enzoz hydrol (68 g/l of ethanol). Potential S. cerevisiae during growth in
optimized media in 150 liter fermenter studies indicated that molasses supplemented with
ammonium sulfate supported 1.5-fold higher specific productivity than that by
unoptimized medium and that in 150 liter fermenter aeration and stirring enhanced
enzyme titre by 1.55-fold over optimized media in 23 litre fermenter. Furthermore, the
cell mass productivity (0.34 g/l h) was 1.33- fold and substrate consumption rate (6.3
g/l/h) was 1.66-fold higher than those in the shake flask. The influence of treatments on
all fermentation attributes of ethanol production was highly significant except for q/S,
which was quite non-significant. The values of the kinetic parameters obtained for
ethanol are higher than the values reported by other workers on the same strain.
xxx
CHAPTER 1
INTRODUCTION
1.1
GENERAL
This collaborative work of Mehran University of Engineering and Technology

Jamshoro and Pakistan Atomic Energy Commission was carried out. A commercial
yeast strain S. cerevisiae was mutated by Gamma irradiation by employing two
approaches obtaining the desired phenotypes of the desired genotypes and the mutant
derivative and then finally selected strain was designated as M-9.Enhanced
production of ethanol, i.e.; 7.5 % (w/v), 95.4 % (w/w) of the theoretical yield and 9.4
(w/v) cell mass and consumed almost all sugars i.e.; 98.6% (w/v) at elevated
temperatures at optimized parameters in fermenters at laboratory and semi
commercial scale bioreactors, digitally controlled through microprocessors.
The research work is presented in four chapters in this thesis. The first chapter
commences with an introduction to the production of ethanol using different types of
raw materials. Detailed description of the fermentation process by yeast, S. cerevisiae,
is given and the effects of different variables on the production of ethanol are also
described in this introductory chapter. Chapter two provides a detailed literature
review and background to the present work. Chapter three describes the material and
methods used to produce ethanol under different conditions. Effects of various
parameters on the fermentation process are highlighted in this chapter. Chapter four
describes the results obtained in this study and discusses the importance of the data in
a wider context. The Final chapter provides the overall conclusion of this work.
1
1.2
ETHANOL AND ITS SCOPE
According to Jeremy (2001), biofuels have the potential to meet the future energy
demands because they are truly renewable energy sources and can be produced
anywhere plants can grow. They are not intermittent and can potentially supply liquid
fuels to the transport sector without major modifications to the existing infrastructure.
Von Sivers et al. (1994) and Wheals et al. (1999) said that ethanol is an important
industrial chemical with emerging potential to be used as biofuel and replace
vanishing fossil fuels. Ethanol (or ethyl alcohol) has been described as one of the
most exotic synthetic oxygen-containing organic chemicals because of its unique
combination of properties as a solvent, a germicide, a beverage, an antifreeze, a fuel, a
depressant, and especially because of its versatility as a chemical intermediate for
other organic compounds.
Ethanol proves itself as a volatile, flammable, clear and color less liquid in normal
conditions. It has pleasant order and suitable taste when diluted with water. The
hydroxyl group is the basis for physical and chemical properties of ethanol (Table
1.1). The group imparts polarity to the molecule and raises the intermolecular
hydrogen bonding. In the liquid state, hydrogen bonds are formed by the attraction of
the hydroxyl hydrogen of one molecule and the hydroxyl oxygen of a second
molecule. This bonding liquefies ethyl alcohol, otherwise it was not possible. The
behavior is similar to that of water in which intermolecular hydrogen bonding is very
strong that water appears to exist in liquid clusters of more than two molecules.
The reactions of dehydration, dehydrogenation, oxidation, and esterification occur
because of the hydroxyl group in ethanol. The hydrogen atom of the hydroxyl group
can be replaced by an active metal, such as sodium, potassium and calcium to form a
metal ethoxide (ethylate) with the evolution of hydrogen gas.
Table 1.1: Important physical properties of ethanol

Property
Value
Normal boiling point, C
78.32
Density, d420, g/ml
243.1
Heat of combustion at 25C, J/g
0.7893
Critical temperature, C
793.0
Lower, vol%
4.3
Upper, vol%
19.0
Gong (1999) is of the view that most of ethanol produced in the world today is starch
or sucrose derived. Van Hoek et al. (1998) said that carbohydrates are readily
hydrolyzed by enzymes, and Saccharomyces cerevisiae easily ferments the resulting
sugars (glucose and fructose) to high concentrations of ethanol.
Costello and Chum (1998) proved that ethanol is a clean burning fuel. Its oxygen
contents decrease emissions of pollutant gasses when combusted with gasoline, and
because ethanol is derived originally from plant matrix, therefore its use does not
contribute to the net accumulation of carbon dioxide in the atmosphere, when used as
fuel. Therefore, ethanol blends have been available for over 20 years at about 30% in
gasoline. It was offered by Wheals et al (1999) in a thorough appraisal of literature
and reported ethanol is environmental friendly, as it reduces pollution and green
house gas emission. It has a positive effect on subsurface soils and ground water and
its falls into sustainable bio products. Ethanol can be formulated from C6 sugars as
under:
(1.1)
The maximum weight % ethanol from the process would be 92/180 = 51.11% about
50% glucose [88/180 (49%)] is converted to carbon dioxide. Hemicellulose is made
up of the C5 sugar (xylose) arranged in chains with other minor C5 sugars interspersed
as side chains. Just as with cellulose, the hemicellulose can be extracted from the
plant material and treated to release xylose which would be converted into ethanol.
1.3
ETHANOL PRODUCTION
Jones (1989) viewed that ethanol can be synthesized, by direct fermentation of sugars,
or from other carbohydrates that can be converted in to sugars, such as starch and
cellulose.
The ethanol can be prepared be ethylene. In the first step, the hydrocarbon feedstock
containing 35-95% ethylene is exposed to 95-98% sulfuric acid in a column reactor to
form mono- and diethyl sulfate:
CH2CH2 + H2SO4 = CH3CH2OSO3H
(1.2)
2(CH2CH2) + H2SO4 = (CH3CH2O)2SO2
(1.3)
Then hydrolyzed with water to give 50-60% aqueous sulfuric acid solution:
CH3CH2OSO3H + H2O = 2 CH3CH2OH + H2SO4 (1.4)
(CH3CH2O)2SO2 + 2 H2O =2 CH3CH2OH + H2SO4 (1.5)
Then ethanol and dilute H2SO4 are separated and in last concentrated sulfuric acid is
formed and recycled. Other processes to make ethanol synthetically are not
commercially important.
1.4
RAW MATERIALS FOR ETHANOL PRODUCTION
Zaldivar et al. (2001) reported that there are three major categories of agricultural raw
materials: simple sugars, starch and cellulose
(Table 1.2)
Table 1.2: Major substrates for fermentative production of ethanol

Sugars
Starch
Cellulose and hemi cellulose
Sugarcane
Grains
Wood
Sugar beet
Potatoes
Agricultural residues
Molasses
Root crops
Municipal solid wastes
Fruit
Waste papers, Crop residue
Heinisch and Hollenberg (1993) have summarized the characteristics and documented
the various aspects related to the growth behavior of S. cerevisiae used in the brewing
and baking industry.
This yeast has been extensively studied and applied widely both in the laboratory and
industry.
1.5
FERMENTATION BY YEAST S. CEREVISIAE
It is believed that yeast S. cerevisiae is very commonly used for ethanol production in
the world (Zaldivar et al. 2001 and Kaisa et al 2006).Some researchers (Nevoigt and
Stahl 1996) have used this strain as rich model strain and its shear stress for have
chosen the yeast strain S. cerevisiae for use as the model aerobic organism in the
experiments mentioning some reason as it is intensive to shear stress, best for food
and beverages, having simple metabolism.
Reed and Nagodawithana (1991) proved that the engineered yeast strains of S.
cerevisiae exhibited a higher fermentation rate than the wild strains. In the absence of
aeration, yeast has the ability to instantaneously change its respiratory metabolism
from oxidative to fermentative one. This catabolic shift is referred to as the Pasteur
effect. It is manufactured by large scale aerobic fermentation of selected strain of S.
cerevisiae. Aerobic growth of S. cerevisiae on fermentable sugars has been studied
mainly in batch culture experiments. The growth characteristics of S. cerevisiae are
variable depending on the condition to which yeast cells are subjected.
Many researchers have studied the factors affecting the growth patterns of S.
cerevisiae under aerobic conditions (Reed and Nagodawithana 1991). Subsequently,
studies in applications of genetic engineering techniques have become very popular
due to the increasing demands of the industry to improve the strains of yeasts. Control
strategies in industrial aerobic fermentation have been developed to maximize the
growth of yeast and minimize the detrimental factors affecting the yeasts growth
patterns.
1.6
AERATION IN FERMENTATION
Pim et al. (1998) revealed that an amount of Oxygen is supplied to the

microorganisms and uniformity could be maintained by agitation. Both parameters are
important in promoting effective mass transfer to liquid medium in the fermenter. The
main function of a properly designed bioreactor is to provide a controlled
environment in order to achieve the optimal growth and product formation in the
particular cell system employed. In laboratory shake flasks, aeration and agitation are
accomplished by the rotary or reciprocating action of the shaker apparatus. Pim et al
(1998) utilized the air stream with the flow rate of 0.5 liter min1.
1.7
OXYGEN TRANSFER
Oxygen must be supplied as per demand of the microorganism for satisfactory growth
rate. That required oxygen will be transfer through the air intake in the bioreactors, by
bubbles present in the reactor. That must be supplied in any mode of the reactor,
batch, semi-continuous or continuous (Doran 1995).The Charles and Wilson (1994)
revealed that separate calculating of coefficient of mass transfer, KL and a is difficult,
but some times impossible.KLa is coefficient of mass transfer instead of KL,which is
directly proportional to the driving force and the area for the air treanfer.That may me
presented mathematically as:
Oxygen Transfer Rate (OTR) = KLa C
(1.6)
and
OTR = KL a (C*L- CL )
(1.7)
Further research was made by other workers too such as Ahmad et al. (1994) and
found an enhanced traditional oxygen transfer rate as the speed of agitator raised
(from 300-600 rpm). Greater agitation produces more dispersion hence the greater
mass transfer rate.Kaster et al. (1990) found that more dispersion could be created in
low agitation if bubble dispersion is utilized for the purpose. If smaller sized bubbles
incorporated then it permits more oxygen and consumes more time to dissolve.
1.8
DETERMINATION OF KLa VALUE
Finding KLa in bioreactors is an important aspect with respect to aeration efficiency by

using many techniques to find the rate of oxygen transfer (Klekner 1988) while
keeping that a system of aeration and homogenization be used, construction of the
fermentation and physiological impact of microorganisms and fermentation medium
composition.
1.9
WHY THERMOTOLERANT YEAST IS USED?
Heat is generated in alcohol fermentation at around 140 cal/g of glucose and would
not be possible for the microorganisms to tolerate it and would result poor alcoholic
yiled.That is to be kept under control through cooling systems, an extra load on the
industry. This proves an advantageous, if heat tolerant yeast is utilized for the same.
That leads to economic production of ethanol. As the industry uses non-amylolytic
and non-cellulytic strain there for starchy and cellulosic substrates need to be
converted into simple sugars. The starchy produce maltose glucose fructose the
cellulosic substrates give xylose, arabinose, glucose, mannose and galactose.
1.10
GENERAL MODIFICATION OF STRAIN
Bailey (1991) and Stephanopoulos and Vallino (1991) reported that general
modification and improvement yeast strain is found important and it is relied on
random mutagenesis or traditional breeding and crossing of strains by screening it out
these techniques provides more properties in the strains. Recombinant technology
enhanced more characterized microorganisms by manipulation was done and
achieved more directed approach. An advancement was recorded when Goffeau et al.
(1996) improved the cellular properties and engineered it by analysis of the cells was
made to identify the most promising targets for the genetic manipulation.
1.11
OBJECTIVES OF PRESENT RESEARCH
Following are the main objectives of the current research:

i.
Development of a mutated S. cerevisae strain tolerant of deoxy-Dglucose .
ii.
Comparative study Native and mutated strain in a fermenter of 23 liter

capacity (15 liter working capacity) at standard conditions.
iii.
Effectiveness of air flow rate on production of alcohol by S. cerevisae

mutant culture.
iv.
Effectiveness of agitation, using mutant cells.
v.
Optimizing the sugar % in substrate concentration in molasses and

hydrol to support maximum product formation.
vi.
Effectiveness of temperature on ethanol manufacturing.
vii. Study of influence of nitrogen source on ethanol production in a

fermenter of 15 liter working capacity.
viii. Effect of different additives on ethanol yield by both wild and mutant
culture in a fermenter of 15 liter working capacity.
ix.
Comparative study of wild and mutated cultures for cell mass and
product formation under optimized conditions in laboratory, semi
commercial (150 liters) scale reactors.
10
CHAPTER 2
LITERATURE REVIEW
2.1 ETHANOL AND ITS BY-PRODUCTS
Sheikh and Berry (1980) isolated thermotolerant yeast in multiple stages, which grows
on molasses and urea medium. This yields a biomass at 30-41 % at 40 oC. Four of
these strains were tested and found resistant on 55 oC when incubated for 15 minutes
time.
Neelam and Amarjit (1991) utilized over ripped grapes and isolated 6 thermal
resistant strains. These heat resistant mutant strains were isolated at 37 oC, by dilution
techniques in yeast extract medium, when irradiated by UV treatment and ethanol
enhanced quantity of ethanol was yileded.Batch reactor was employed and using 20
% total sugars. Iconomou et al. (1991) also enhanced ethanol production form the the
molasses fermentation medium using gamma-rays
Argiriou et al. (1992) revealed that 17.6 % and 16.5 % alcohol may be obtained from
the two strains of S.cereviae, named as AXAZ-1 and AXAZ-2,respectivel.Grapes
must was used as a source for the sugar substrate.
Laplace et al. (1992) presented the kinetic behavior of six mutated strains of yeast
species on the medium containing D-galactose.
Christer (1993) grew yeast strains of S.cerevisiae through the technique of metabolic
uncoupling. Carbon and energy sources were used for that chemostat culture.
Gardner (1993) presented a study of 14 strains of S. cerevisiae and determined the
growth pattern of these microorganisms by using a variance of 100 to 300 ppm for
glycerol production through a fermentation process.
11
Roukas (1994) revealed the results of the kinetics of ethanol in shake flask
fermentation experiments through S. cerevisiae and presented ethanol yield for 3.56.5 % .on the temperature range of 30-35 oC.
Sonia and Miguel (1994) analyzed the glycolytic flux of yeast S.cerevisiea and
calculated the production rate in chemostat culture and found out coefficients of
metabolic concentration.
Win et al. (1996) presented a study regarding an experiments base upon the yeast S.
cerevisiae by cassava starch syrup and molasses medium in batch fermentation.
Yadev et al. (1996) has isolated yeast, named, HAU-1 on molasses medium in a
reactor, containing columns. That yeast was utilized and found the ratio between
length and diameter o f the reactor. It was asserted that it showed lower efficiency of
that reactor but could be enhanced through using supplements of nutrients.
Banat et al. (1998) reported that the heat resistant yeast of sugarcane molasses is able
to work at a temperature more than 40 C.
De et al. (1998) presented a model fermentation in which biomass, sugar, ethanol,
diacetyl and ethyl acetate are taken into account and all other parameters were also
monitored
Pim et al. (1998) presented a study stae growth rate of at industrial fermentation to
find out the dilution rate, respiration in the system was kept under the study for
ethanol production.
Sheoran et al. (1998) reported an active cell and optimized the rate fof production
through the yeast strain UUA-I in vertical column reactor. It was revealed that the
yeast cells are 30 % active in that reactor and are capable for ethanol fermentation, if
yeast beads were employed in the reactor at 40 oC.
12
Domingues et al. (1999) presented a study for the alcohol fermentation through
K.marxianus and S. cerevisiae species of yeast. An expression was done for LAC12
and LAC4 (lactose permease and (-galactosidase).Ethanol production rate was
recorded as an increased one at seven times and found that the system is stable for
long six month period time.
Newman et al. (1999) worked for the data of a Parental Stress Index [PSI] for the
yeast stress release factor in heat shocked proteins (Hsp104) and it was pointed out
that defense mechanism of yeast release factor Sup35.
A study was carried out for two genes, MIG1 and GAL80, for the utilization of
galactose by Ronnow et al. (1999) for an industrial strain for ethanol distillery.
Physiological characteristics were investigated on mixtures of glucose and galactose
and on molasses for the same.
Abdel-fattah et al. (2000) has reported an enhanced temperature for synthesizing the
Hsp from various microorganisms, during fermentation process
Atiyeh and Dvnjak (2000) used S. cerevisiae ATCC 36858 and beat sugar molasses
medium in batch fermentation utilizing total sugars at 94.9 to 312 g l-1 for ethanol
production at 93 % of the theoretical yield,which was lowered with the lower % age
of total sugars
Sreenath and Jeffries (2000) experienced 43 forest products laboratory (FPL) strains
of Pichia stipitis and Candida Shehatae for their ability to ferment a 1:1 mixture of
glucose and xylose to ethanol prior to fermentation of partially deacidified wood
hydrolyzates. The starting sugar composition, pH, and concentrations of inhibitors
such as acetic acid, furfural, and hydroxyl methyl furfural varied from one batch to
13
another. The delay observed in growth and fermentation depended on the amounts of
inhibitors present and on the capacity of the strain to resist them.
Gimenes et al. (2002) determined xylose concentrations in a shake flask experiment
and significant growth was recorded at increase values of oxygen intake.
Carvalho et al. (2003) has worked on ethanol production through the yeast S.
cerevisiae grown on molasses medium in a fed batch culture system. All fermenter
parameters and the kinetics may also be presented for yields, inoculum, substrate
consumption and inhibition rates.
Alfenore et al. (2004) presented an optimized strategy for aeration rate in the bio
ethanol production. Aeration conditions were also quantified and showed a high
performance for the S.cerevisiae cell
De Neto et al. (2004) screened out some non-flocculating type of yeasts growth
factors for the yeasts, other than the yeasts during grapes juice fermentation medium.
It was also studied that what does it effects if the concentration is at very low one. The
morphologivcl study was the another parameters for this study.
Najafpour et al. (2004) carried out a successful fermentation study of total sugars
concentration consumptions by S. cerevisiae for ethanol productivity through an
immobilized reactor. For its long 24 h operation time.
Rajoka et al. (2004) has investigated the outcomes of carbon resources and its
attentiveness, and different fermentation parameters and their effects on the
production of beta-glucosidase through a high temperature resistant K.marxianus at
shake flasks level.
Marchetti et al. (2005) presented advantages and disadvantages of alternative
technologies for the use of biofuels production. Methanol, Ethanol and Butanol were
14
presented. Sodium hydroxide, potassium hydroxide, sulfuric acid and supercritical

fluids and heterogeneous ones such as lipases were used as catalyst.
Rajoka et al. (2005) mutated and thermotolerant S. cerevisiae ATCC 26602 ,through
multiple screening techniques by the use of UV radioactivity, which was made
possible to work at 40 oC and produce enhanced production of ethanol at 1.6 folds.
Shang et al. (2006) developed a laboratory scale bioreactor of 5 l volume for the yeast
culture at high cell density and other keeping other reactor parameters under control.
He revealed that the feed rate of glucose was adjusted with the ethanol concentration.
Other reactor components were maintained at these values: Temperature, 30 oC, pH
5.5, agitation, 300rpm and fermentation retention time 60 h, while respiration was
kept at 1.0 and ethanol concentration at 1 %.
Jurascik et al. (2006) offered a metabolic pathway model and used a modified
equation of Monod. Found all kinetic parameters keeping growth rate proportional to
enzyme concentration. Three routes for the yeast S.cerevisiae 424A (LNH-ST) were
experienced for glucose and xylose fermentation as: lactose and ethanol oxidation
reduction of lactose, with sugars concentration at 20 g l-1
Muenduen et al. (2006) used flocculating yeast, S. cerevisiae M30 and cane molasses
as a substrate. 12 kinetic parameters for ethanol production, cell mass growth and
sugar consumption were found and temperature effects were recorded in this research.
Activation energy, death rate and
Arrhenius plot.
ethanol production rate were correlated with
15
2.2
YEAST AND INVERTASES
A conversion takes place through -Fructo furanosidase (EC 3.2.1.26) in which

sucrose is converted into fructose and glucose. Most of food and pharmaceutical
industries utilize this enzyme. The enzyme also possesses fructosyl- transferase
activity and can lead to formation of fructo-oligosaccharides which have achieved
great attention because of several favourable properties for health foods (Hayashi et
al. 1992; Roberfoid 1993; Tomamatau 1994; Yun 1998). A number of cultures make
this type of enzyme.(Hayashi et al. 1992; Euzenat et al. 1997; Muramatsu &
Nakakuki 1995; Roberfoid 1993; Yun 1998).
It is very important to screen organisms with the help of sucrose as an inducer for the
enhanced production of enzyme at commercial scale. (Hayashi et al. 1992). In our
country, sucrose is needed as sweetener for human consumption and there is no
surplus sucrose to be utilized for production of invertase. Its production from
molasses could improve economics of -fructo-furanosidase (Ffase) production.
Sugarcane molasses contains may contain up to 25-40% glucose and fructose which
exert catabolic repression on Ffase production (Rincon et al. 2001)Enzymatic
manufacture of enzymes is prejudiced with the help of insertion and synthesize
through catabolite (de Groot et al. 2003). Carbon catabolite repression alters with the
help of protein, named as CreA (de Vries et al. 1999). Sucrose is Ffase inducer and
liberates sugars and not feasible for Cre A structure (Hrmova et al. 1991and deVries
et al. 1999).Fungi is also regulated in the same pattern (deGroot et al. 2003; deVries
et al. 1999 )
16
Saccharomyces cerevisiae produces both extracellular and intracellular -fructofuranosidase in submerged fermentation (Rincon et al.2001). Enhancement in the
enzymatic expression of Ffase increases substrate consumption allows for permease
(Rincon et al. 2001). Isolation of glucoses is regulated through mutants. (Rajoka et al.
1998; Haq et al. 2001). The separation of this strain is improved and beneficial.
Kaiser et al. (1986) constructed a series of indicators of the enzyme invertase. Agudo
and Zimmermann (1994) observed a low level invertase activity. Vitolo et al. (1995)
permitted this strain to grow through molasses by variation of parameters like DO,pH
and sugar consumption rate.
Sturm (1996) presented observations for the invertases hydrolyzation from sucrose
into glucose and fructose. Zhu et al. (1997) created relationship among activity and
concentration. Niuris et al. (2000) articulated this product and presented its properties.
Tanaka et al. (2000) experimental shows that the product is higher in quality form the
native cells. Ghosh et al. (2001) has purified the invertases and produced high quality
of it. Niuris et al. (2000) has produced a wide range of microorganisms by utilizing
nutrients. Maria et al. (2002) purified production of invertases by using a variety of
nutrients through SDS-PAGE. Rossi et al. (2003) worked on the entrapped cells
grown and shown their growth patterns and also found that they are consuming more
sugars.
17
2.3
OTHER PARAMETERS
2.3.1 Substrate concentration

Sivaraman et al. (1994) reported a high yield at high consumption. Myers et al. (1997)
considered action this thermotolerant yeast and found a high consumption of substrate
and have a direct relationship with HSuc for example bread particles.
2.3.2 Nitrogen and carbon sources

Najafpour et al. (2004) is of the opinion that reported CSL is the best source for this
thermal resistant yeast. It was also found that a reasonable % of ethanol was also
yielded up to 12.5 %.However Sues et al. (2004) found (NH4)2SO4 preferable sources
forth Nitrogen.
2.3.3 Airflow rate

An airflow rate of 0.5 v/v was utilized by Pim et al. (1998) and reported a 60 % of DO
from 800 rpm using smaller bioreactor of 2 liter size.
2.3.4 Additives
Tween 80 was utilized by Castro et al. (1995) and Dragone et al. (2003) as additives
for the fermentation and uphold the ethanol production rates,putting notes that there
was a more time required in this experimental work. Gasch et al. (2000) have used
tween 80 and found that it could possibly be transferred at larger scale production and
concluded that the experiments are that of laboratory scale and need further study at
fermenter and above scales. Vitamins are also experienced by Alfenore et al. (2002)
as additives in high yield ethanol with a disadvantage that glycerol is produced as an
18
additional production which reduces the ethanol production rate. Reddy and Reddy
(2005) experience more time in high yield of ethanol when additives are utilized
during the fermentation process.
2.3.5 Thermodynamics of ethanol and Ffase formation

Many activities are recoded when a cell goes under a metabolic pathway by which an
unordered molecule will under go some changes through a catalyst. An experimental
data was presented by (Agarwal 2006) for the movement and reaction rate. (HammesSchiffer 2002). Very enhanced production of enzymes could be possibly achieved
with the conditions if they are bound the transition state energy. (Eisenmesser et al.
2005). Garcia-Viloca et al. (2004) have worked for the new developed theory for
transition energy for all types of energies that will confirm fluctuated, active effects
for improved enzymatic catalysis. Garcia-Viloca et al. (2004) and Eisenmesser et al.
(2005) have presented almost similar findings for the electrostatic that leads for
entropy. Agarwal (2006) suggested a functionality of enzyme catalysts which is
effective for enhancement in proteins for getting promoters and cross behavior with
the help of current researchers.
Wolfenden and Snider (2001) observed that enzymatic catalysts can expedite the
reactivity of the chemicals at a range of 100-1000 s
. Siddiqui et al (2002) have
recognized proteins, which are capable in a flexible structure and increase the
coefficients of activity, kcat .
Brisol et al. 1999; Heijen (1999) reveled that in the processing of microorganisms
there are three basic interactive variables; biomass, substrate and product which may
keep the coefficients of maintainability stable ones and helps in finding the rate of
19
productivity. Stephanopoulos et al. (1998) and Maskow and Stockar (2005) have
provided a useful information regarding biological and thermodynamic processes and
their metabolic reactivities and silico predictions. (Goldberg et al. (2004) have applied
the thermodynamic data to industrialized systems for direct calculations of
stiochiometry in nature and are providing assistance to engineers for the energy needs
of the plants.
Garcia-Ochoa et al. (2000) presented a study for an optimized control of the
bioprocess plant .They proved that it requires a model for the various kinetic
parameters too the reason behind is to calculate the stability of the culture and control
of the bioprocess. Liu et al. (2003) have presented an empirical formula, the Monod
equation for the thermodynamic properties the fluid That was used for the study for
the various mediums in their viscosity would possible effect the rheological
characteristics of a fluid issues in materials shifting and lowers the activity of
metabolic conditions and used for the optimizations of the transfer of oxygen and
their rates of aeration and agitation for getting the fluid mixed. Thermal motions
create an enhancement in the reactions of enzymes and rate of transition (Fisher
2005). When the temperature effects on the production it enhances the state of
transition and increase the rate of formation in that. (Benkovic and Hammes-Schiffer
2003). The Monod equation for the kinetic changes and modifications is used by
Kelly (2004) and the research is made for the consuming the substrate .in it. And mass
formation for these cells under the study.
Roels (1983) has explained the way among the three, which are now introduced for
the finding out the thermodynamic values in any enzymatic system. This was also
found useful for the time dependant variables in the reversible equations there. Tow
20
different models of transitional state and Arrhenius theory was joint together by Aiba
(1973) and that was found successful in the systems where thermodynamic and
transitional state is used. However previous workers (Arni et al. 1997) have used the
thermodynamic and transitional parameters in the fermentation systems for the
bioproducts. Arni et al. (1999); Converti and Dominguez (2001) have applied values
for the production and the enzymes ocncentratiuon rate in kinetic thermodynamic
parameters of the reactions .This has been followed by other workers too(Converti
and Del Borghi 1997; Converti and Dominguez 2001; Rajoka et al. 2003).
Rate of change in the consumption depends on the value of the coefficient of transfer
that is equal to the identity (Day et al. 2002)This was proved by the current research
and is mostly applied to the media which is less visoucs (Garcia-Viloca et al.
2004)applicable to non-viscous media. (Agarwal 2006). Ln (A) is also having same
value for the determination (Winzor et al. 2005).
Enzymatic reaction is reversible when folding and unfolding of the enthalpy proteins
are considered. (Beadle et al. 1999; Shiraki et al. 2001).A week interaction creates a
unstable or stable due to entropy which is conformational one. It shows that the
difference e is very small in the entropies. The stability is dependent of the product
formation (Eisenmesser et al. 2005). A reasonable number of the proteins is formed
when the cells are shocked by the heats or the therms and these proteins (Borges and
Ramos 2005).
High rate of enzymatic production could be achieved, if and when the enthalpy
changes with respect to the substrate changes. This is also useful for the contributing
enzymes there. This study was made to make some results on the basis of temperature
profile for the reactions and the systems (Winzor et al. 2005).
21
Agarwal (2006) has freshly recommended that protein ambiance is very important for
finding out the way to bimolecular process to get a rid from the local energy barriers
for the bioprocesses. That will solve the problems of energy at all. That has a very
strong effect on the forming a production and to modify the proteins very efficiently
and rapidly in a fermentation process.
22
CHAPTER 3
MATERIALS AND METHODS
3.1
RESEARCH CENTERS
This thesis was conducted in a close collaboration with the National Institute for
Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. This research
institute is under the administration and management of Pakistan Atomic Energy
Commission (PAEC), Islamabad. The added benefit of this research collaboration has
led to the establishment of an advanced Biochemical Engineering Laboratory in the
Chemical Engineering Department, Mehran University, Jamshoro. It is anticipated
that this laboratory will be ready for project students and advanced research work in
near future.
3.2
SAMPLE CENTERS
The substrates, black strap sugar cane were taken from sugar industries of various
parts of Pakistan.
3.3
MICROBIAL STRAIN
Yeast strain of S. cerevisiae SAF (France) was purchased from a local market and
grown at the most popular yeast medium at minimum composition of the constituents
as shown in Tables 3.1 and 3.2 as used by Rajoka et al. (2005). Then the culture was
further stabilized (through mutagenesis) for higher working temperature and
catabolite repression resistant at the same time to make it thermotolerant with
retention of hyper-production of ethanol power and used for enhanced ethanol
production.
23
3.4
MAINTENANCE OF CULTURE
The strain was maintained according to Sirianuntapiboon et al. (2004) on yeast
growth media (Fig. 3.1). Different chemicals of analytical grade were used for
preparation of the yeast growth medium. The chemicals were added into distilled
MUTANT CULTURE OF
flask of 500 ml capacity. Through the solutions of Hydrochloric acid (1 N) and
KLUYVEROMYCES
MARXIANUS
water one by one with shaking and volume was made upto 100 ml in an Erlenmeyer
Sodium Hydroxide (1 N) the media pH was kept upto 5.5.
Figure 3.1:
Plate culture of fresh thermotolerant S.cerevisiea
14
24
3.5
GROWTH MEDIUM COMPOSITION
Table 3.1: Chemical composition of growth medium

Chemicals/ Biochemicals
w/v (%)
Ammonium sulfate
0.003%
Potassium di hydrogen phosphate
0.001%
MgSO4
0.005%
KCl
0.005%
Yeast extract
0.5%
Malt extract
1.0%
Glucose
1.0%
Peptone
1.0%
Agar
2.5%
All of the chemicals and biochemicals were that of quality and purchase d from well
reputed companies.All chemicals were of analytical grade and were purchased from
Sigma/Aldrich Chemical Company, Oxoid Chemicals (USA), Sharlau, (Spain) and
Merck Co. Germany.
3.6
PREPARATION OF PLATES
Distilled water was dispensed through 500 ml Erlenmeyer flask and the chemicals
required, as described earlier for preparation of the yeast growth media, were added
one by one with shaking. Then the volume was maintained upto 100 mls in a conical
flask for optimization studies or pH was varied for pH optimization studies. This well
plugged and covered with aluminum foil was sterilized in an autoclave machine at a
prescribed machine parameters i.e.,121C, 15 psi for 10 min. After autoclaving, the
yeast growth medium was poured into the Petri plates. Upon solidification of media,
25
the plates were kept at room temperature for one day to confirm the purity. Later on,
Petri plates were inoculated with S.cerevisiae and then by streaked and incubated at a
temperature of 37 C for 24 h. When the colonies were formed, the plates were
properly sealed by parafilm then preserved for the long time at lower temperature of 4
C.
3.7
PREPARATION OF SLANTS
The yeast growth media, prepared by the procedure already discussed in section 3.6,
were equally distributed in 10 test tubes (Pyrex) properly cotton-plugged and covered
with aluminum foil. The tubes were autoclaved at 121 C, 15 psi for 10 min. The
autoclaved tubes were laid down in slanting position for some time for solidification
of media. The slants were inoculated with S. cerevisiae by streaking, and then
incubating at the temperature of 37 C for 24 h.
3.8
PREPARATION OF THE CULTURE OF NATIVE S. CEREVISIAE
3.8.1 Preparation of yeast growth medium

Distilled water (50 ml) was dispensed in an Erlenmeyer flask of 250 ml capacity, and
following chemicals were added and the pH was kept as in section 3.8.
Table 3.2: Chemical composition of inoculum medium
Chemicals/Biochemicals
w/v (%)
Ammonium sulfate
0.05
Potassium di hydrogen phosphate
0.05
Magnesium sulfate
0.05
Yeast extract
0.5
Glucose
2.0
pH
5.5
The pH of the inoculum was kept 5.5 as described in the section 3.4
26
I. A well grown single colony of S. cerevisiae was picked up by a loop and was
inoculated in 100 ml of yeast inoculum medium in a conical flask was
incubated in a shaking incubator (Toshiba, Japan) at 37 C at 120 rpm for a
period of 24 h.
II. Properly diluted culture (100 l) was taken after 24 h of growth and spread on
the yeast media plate for incubation as described in section 3.8 part I and
stored as described previously for further utilization.
Yeast growth medium was prepared with the help of biochemical and salts as
mentioned in the table 3.2 and as described before (section 3.8) for the required
volumes and autoclaved as per described method in section 3.4 for 15 minutes. These
sterilized and cooled cells were grown after inoculation and then centrifuged as
mentioned
in
the
previous
section
3.4
and
3.5
and
analyzed
through
Spectrophotometer (LaboMed USA).

A known volume (50 ml) dispensed in a 250 ml Erlenmeyer flask; the chemicals,
with the composition already mentioned in Table 3.2, were added to prepare the yeast
growth media. The pH of the medium maintained as described in section 3.8.
I. Yeast inoculum medium was inoculated with a loopful of purified culture of S.
cerevisiae aseptically in a laminar hood and then the flask was incubated at 30
C at 120 rpm in orbital shaker for 24 h.
II. After growth. 10 ml of culture of S. cerevisiae was taken aseptically in the
autoclaved McCartney vials of 30 ml capacity.
III. McCartney vials were labeled for exposure of different doses of gamma
radiation i.e. (0.20 to 1.00 kGy in the span of .20 kGy ).
27
IV. They were properly sealed with parafilm and packed in polyethylene packets
to avoid the leakage and contamination of culture of S. cerevisiae with water
in the tank during gamma radiation exposure.
V. After exposure of culture of S. cerevisiae to different doses of Gamma
radiations. McCartney vials were stored at 4 C for further usage.
3.9
EFFECT OF GAMMA RAYS IRRADIATION ON VIABILITY OF

CELLS
The untreated culture of S. cerevisiae was taken as control. The 100 l of native and
gamma rays irradiated cultures (0.2, 0.4, 0.6, 0.8 and 1.00 kGy) of S. cerevisiae were
diluted in 900 l of biological saline to make 10 times dilution. The cultures were
7
further diluted upto 10 and were spread on growth plates separately by spreader
aseptically. The plates were labeled and incubated at 37 C. After growth of 24 h, the
viable colonies were counted and colony forming units/ml (CFU/ml) were determined
as follows:
Example:
Viable Counts
= 1324
Colony Forming Unit (CFU)/ml
=Viable countsx1
Sample volume x dilution factor
= 1324 x 1
7
0.1 x 10
11
=1.324x10
Cells /ml
3.10
SELECTION OF MUTANT OF S. CEREVISIAE
The survival curve was prepared to select an exposure dosage for mutation, giving 3log Kill (Rajoka et al. 1988) and exposure dose of 1.0 kGray (kGy) giving 3-log kill
(0.1 % survival).Two strategies used for mutant selection simultaneously. In first
strategy the mutant was selected by directly spreading the irradiated cells on plates
28
containing DG 1.5 % (w/v), sugars 17% (w/v) in yeast medium and agar 2.5 % (w/v).
In second strategy, the mutants were selected by permitting irradiated cells to express
in broth containing 17% sugars plus 1.5 % deoxyglucose at 45 oC until the OD
reached 0.1 at 10 dilution and plated on PDA plates containing 1.5 % deoxyglucose
and incubated at 45 C .The mutants were grown at different temperature (37, 40, 43,
45 and 47 C) on yeast agar plates. The best mutant of S. cerevisiae was selected on
the basis of its thermotolerance.
Similarly the mutants were grown in yeast fermentation medium at different
temperatures (20, 22, 24, 26, 28, 30, 35, 40, 43, 45 and 47 C) and best mutant of
S.cerevisiae was selected on the basis of thermotolerance.
3.11
PURIFICATION OF MUTANTS OF S. CEREVISIAE:
The mutant cells of S. cerevisiae were purified in the same way as mentioned for
native strain with the exception that mutants were grown at 45 C. Later on plates
were inoculated by S. cerevisiae by streaking, and incubation was done as in section
3.4 to 3.6 and preserved for next experimentation after sealed properly by parafilm
and stored at 4 C.
3.11.1 Slants of purified culture
The yeast growth medium, prepared by the procedure already discussed, was
equally distributed in 10 test tubes (Pyrex) which were properly cotton-plugged and
covered with aluminum foil. The tubes were autoclaved as before and the autoclaved
tubes were laid down in slanting position for some time for the solidification of
media. The slants were inoculated as earlier.
29
3.12
PROPAGATION OF YEAST
The yeast was propagated for 23 liter bioreactors. Then the yeast was propagated for
semi-commercial production in two stages to increase the volume. The naming was
used as the first stage propagation and the second stage propagation.
3.12.1 First stage propagation
At the first stage propagation cell culture was inoculated in a one liter volume flask to
make a volume of 250 mls carrying Di ammonium phosphate, yeast extract along
with glucose ,2.5,2.5 10,2, gl-1 was incubated as mentioned earlier..Fermenter of 23
liter volume (Made in Germany) was used to carry out this research studies. The
operating volume of the reactor was 15 l. All important accessories were attached
with the reactor for pH, DO, aeration, agitation and mixing. The molasses medium
containing 15% or sugar concentrations others in g/l-1) of the nutrients used in flask
experiment such as ( NH4) 2 SO4, 2.5 and yeast extract 2.0 g and pH was adjusted at
5.5 with sulfuric acid. After steam-sterilization for 45 minutes at 121 C for 30 min at
1 bar. Air and circulation water were opened till temperature came down to 50 C.
The reactor media was inoculated with that of ten percent at 30 C. 15 liters per hour
was utilized an air flow rate for initial eight hours that will make a rich production of
cell mass .Then the rate was reduced upto three liters per hour for next fermentation
operation of the reactor. For the process optimization the reactor operation was made
continuous upto 28 hours and the foam was made under control by the use of
antifoam ,Silicon oil .At this stage, the fermentation broth had a viable cell count at
300(106 cell/ml and total sugars measured as brix was 15-19. That was ready to
transfer to stage II when pilot plant studies were performed.
30
Fig. 3.2: A view of the 23 Liter Fermenter
31
Fig. 3.3: A view of a 150 Liter Fermenter
32
3.12.2 Second stage propagation
At this stage the working capacity of the closed vessel made up of stainless steel of
100 l was used. First of all vessel was sterilized as mentioned in section 3.13.1. Then
the yeast inoculum was transferred in these tanks as stated above and the fermentation
started.
3.12.3 Fermentation
Fermentation tanks were made up of stainless steel, having capacity of 23 and 150 l.
All experiments were performed in batch fermentation (Fig.3.2 and Fig. 3.3). The
temperature was maintained at 30 C for optimization studies of process variables.
The mutant was grown at 43-45 C or as mentioned other wise. The temperature was
controlled by cooling water passing around the mash through the jacketed vessel.
Substrates (molasses and Enzoz hydrol) of the optimized brix/concentrations were
used. Level of the fermenter was raised during agitation so one third volume of vessel
was kept void in each study. This circulation ended after 48 h, by continuous adding
of silicon oil as an antifoaming agent. Fermented mash from the fermenters was
sampled at every four h. A Brix hydrometer utilized for checking the specific and was
confirmed on HPLC. Ethanol (already separated through extractor a t laboratory
scale) was known through HPLC. When molasses was used as a carbon source,
almost 100 % total sugars (TS) were consumed after 24-28 h fermentation. Peaks of
ethanol were observed after a retention time of 19.30 to 19.36 min as mentioned in the
Figure 3.4.
33
3.13
EFFECT OF CARBON AND NITROGEN SOURCES ON ETHANOL

PRODUCTION
Black strap sugar cane molasses was used as a carbon source and compared with corn
molasses (known as Enzoz Hydrol) in the production medium. The effect of different
substrate concentrations i.e. 5, 8, 10, 12, 15, 17.5 and 20 % (w/v) were tested to
determine the optimum substrate concentration for maximum ethanol production by
mutant strain of S. cerevisiae. 0.23 % of nitrogen was utilized from CSL, di
ammonium phosphate (DAP) and Urea for the growth medium as utilized previously
by Favela-Torres et al. (1998) and Gutierrez-Rojas et al. (1995).
3.14
EFFECT OF DIFFERENT ADDITIVES ON ETHANOL YIELD BY

BOTH WILD AND MUTANT CULTURE IN 23 LITER FERMENTER
Tween 80 and NaF were tested as additives for the enhancement of production of
ethanol through S.cerevisiae mutant in the fermenters of 23 liters and then in 150
liters.
3.15
EFFECTIVENESS OF AIR IN ETHANOL PRODUCTION
The air is essential for supporting a basic quantity of cell mass in fermenters. Earlier
studies (Rajoka et al. 2005) suggested that at initial 8 hours air rate may be at 1 v/v
followed by supplying air at slower aeration rate was sufficient for ethanol production
process. Thus the ethanol fermentation was carried out for the producing ethanol
through a thermotolerant yeast S. cerevisiae. As mentioned earlier that the agitation
intensity helps in improving mass transfer of air, production of biomass airoptimization of air transfer rate and agitation rate was performed. During
fermentation, data on cell mass, ethanol, concentration of sugars in mash were
collected for calculation of different process kinetic parameters.
34
3.16
ANALYTICAL METHODS
3.16.1 Preparation of standard curve for biomass estimation

Small volume (50 ml) of cell culture of S. cerevisiae was harvested after incubation of
24 h at 37 C that was separated in a centrifuge, as mentioned in section 3.5 and the
pellets were washed with saline. Then the material was recentrifuged and was dried
on filter paper in hot air oven. The dry cells were grinded to a fine power. Stock
solution-I was prepared by dissolving 100 mg of grinded dry cells in 4 ml distilled
water and stock solution-II was prepared by adding 400 1of stock solution-I to 9.6
ml distilled water. Various dilutions of stock solution-II (upto 10 fold) were made to
make calibration curve. The optical density of each dilution was noted at 610 nm on a
digital Spectrophotometer (Spectro-UV-VISRS, Labo Med. Inc USA). The
absorbance was adjusted to zero with blank, which was distilled water.
3.16.2 Biomass estimation

Effect of different process variables like nitrogen and carbon sources and their
concentrations, additives, fermentation temperatures, media pH, dissolved oxygen, air
flow rate, agitation intensity, was determined during biomass formation. Different
time course samples of native and mutant strains of S. cerevisiae were subjected to
determine optical density at 610 nm on the spectrophotometer after making proper
dilutions.
The absorbance was adjusted to zero with distilled water as blank. The samples were
diluted and centrifuged as mentioned in the section 3.16.2.Optical density (OD) of
diluted cell free solution noted down and subtracted from total OD of the culture
35
broth with cells. The OD was multiplied by slope of the plot of standard curve to get
biomass in mg cells/ml.
3.16.3 Extraction of ethanol
After fermentation, cells were separated through centrifugation operation as
mentioned earlier. Ethanol was distilled with Soxhlett apparatus (Japan) by setting its
temperature at 80 oC. After getting the distilled sample, either volume was measured
or its volume was made up to its original volume with deionized water, filtered (in
filter paper 0.22 microns) and microfuged (7,000 rpm, 3 min). Ethanol concentration
was confirmed on HPLC as mentioned earlier (Rajoka et al. 2005).
3.16.4 Ethanol estimation through HPLC
Distilled and filtered samples of ethanol were run in High Performance Liquid
Chromatograph (HPLC) (Perkin Elmer, United States of America) using column
HPX-87H (300 x 78 mm) (Bio, Richmond, California) maintained at 45C in a
column oven. Sulphuric acid (0.001 N) and HPLC grade water was utilized as a
mobile phase at 0.6 ml/min. The samples were detected by refractive index detector
and quantified using Turbochron 4 software of Perkin Elmer, USA.
Fig. 3.4:HPLC chromatogram of fermented molasses and untreated hydrol

containing 15 % TS
36
3.16.5 Harvesting of intracellular invertases

The intracellular invertases were extracted by sonication from the culture of native
and mutant strains of S. cerevisiae. After growth of the yeast, the culture was
centrifuged and the cell pellets were washed as earlier .The cell mass pellet was
suspended in normal biological saline (0.89 % w/v of Na Cl). The cells of exact mass
were taken for the sonication. They were vortexed to get homogeneous mixing of
cells and were disintegrated by ultrasonic waves (10 sec impulses 5 seconds rest for
20 cycles) in ice (To avoid the denaturation of intra-cellar proteins at high
temperature attained during sonication, ice was used during this operation). After
sonication, the samples were recentrifuged to settle down the disintegrated cell debris.
The supernatant having intracellular invertases was taken and preserved at -20C.
3.16.5.1 Invertase assay
The activity of invertases checked through was determined (by using of 100 l
enzyme 10 m1 McllVain buffer of 0.15 Molality and maintained a pH of 5.5) or 50
ml sodium acetate (pH 5.0) and 1.5 (w/v) sucrose solution was used as substrate. That
mixture was sent for incubation 50 C for 15 minutes a shaking bath. Then quenching
performed through the placement of that reaction mixture in running water for 5 min.
The amount of glucose was determined by adding 100 l of reaction mixture to 1 ml
of glucose oxidase based glucose measuring kit (Biocons, Germany) and was then
incubation
was
done
then
Optical
Density
(O.D.)
was
taken
Spectrophotometer taking a previous method of Hayashi et al. (1992).

3.16.5.2 Determination of units for invertase activity
Glucose concentration in assay was determined using the following equation:
Glucose concentration = A of samp1e conc. of standard (100 mg/dl
through
37
= A of standard
For example:
A of sample
A or standard
Glucose concentration
= 0.12
= 0.039
= 0.12 100 ml
0.039
= 307.69 mg /100 ml = 3.0769 mg /ml
Total volume of reaction mixture
= 2.1 ml
Concentration of glucose in assay
= 2 3.0769 = 6.46149 mg/ml
1 mol of glucose
= 0.1802 mg
Total moles of glucose released by invertase = 6.46149 = 35.86
0.1802
Incubation time {-or invertase activity
= 15 min.
moles of glucose released in 15 min
= 35.86
moles of glucose released / min.
= 35.86
15
= 2.391 mol/min
1000 l of invertase will liberate
= 2.391 1000
100
= 23.91 units
Invertase activity
= 23.91 U/ml/min. under the assay
conditions
3.16.6 Glucose concentration determination
DNS method was used for the purpose of sugars referring previous method used by
Miller (1959)
3.16.6.1 Preparation of DNS (Dinitrosalicylic Acid) solution
Different ingredients were used for the preparation of DNS. These are as follows:
(i)
Distilled water
1416 ml
(ii)
3-5, Dinitrosalicylic acid
10.6 g
(iii)
NaOH
19.5g
The above ingredients were dissolved and gently heated in water bath at about 80C
until a clear solution was obtained. Then the following chemicals were added:
(iv)
Rochelle salt
(Sodium Potassium tartarate)
19.5 g
38
(v)
Phenol (melted at 60C)
7.5 ml
(vi)
Sodium metabisulfate
8.3 g
After dissolving all the above ingredients, the solution was filtered through a large
coarse sintered glass filter and stored at room temperature in an amber bottle to avoid
photo-oxidation. It was stable for 6 months.
A zero point absorbance was adjusted by blank containing 3 ml of distilled water and
3 ml of DNS reagent.
3.16.6.2 Standard curve of glucose
Different known concentrations of 0.1 % glucose was taken and diluted to a final
volume of 3.0 ml with citrate phosphate buffer as shown in Table 3.3
Table 3.3: Concentration of glucose for standard curve
S.No
Concentration of
glucose solution (l)
Distilled
water (l)
Buffer
(ml)
Total
volume
(ml)
DNS
reagent
(ml)
Absorbance at
550 nm
200
800
2.0
3.0
0.235
400
600
2.0
3.0
0.47
600
400
2.0
3.0
0.520
800
200
2.0
3.0
0.690
1000
0.00
2.0
3.0
0.860
Samples were boiled in boiling water bath for 15 min. The reaction was quenched on
ice for 15 min before taking reading on Spectrophotometer as described earlier. Then
it was plotted against different glucose concentration (g/ml) to draw standard curve
using Slidewrite 3 software.
39
3.16.7 Substrate utilization

The substrate utilization by native and mutant strains of S. cerevisiae, during the time
course study was determined by DNS method and glucose measuring kit to analyze
total and reducing sugars in the samples.
Standard
The 10 l of standard solution and 90 1 of distilled water were added to 1 ml glucose
kit incubation was done for 5-10 minutes and then 500 nm on spectrophotometer as
mentioned earlier.
Blank
Known volume (100 1) of distilled water was added to 1 ml glucose kit and the
absorbance adjusted at zero.
Sample
Known volume (100 l) of diluted sample was added to 1 ml glucose kit and OD
recorded after incubation of 5 to 10 minutes at 500 mn of spectrophotometer.
For Example:
A of sample
= 0.61
A of standard
= 0.29
Glucose concentration =0.6l/0.29x100=210.35mg/100 ml
= 210.35 mg/100 ml
= 210.35 (dilution factor
= 210.35 ( 20
= 4207 mg/100 ml
= 4.21 g/100 ml
= 42.1 g/1
3.17 CHEMICAL COMPOSITION OF HYDROL (STARCH MOLASSES)
Properly diluted and filtered hydrol was analyzed by HPLC using RI detector. This
substrate contained
40
Fig. 3.5: HPLC chromatogram of properly diluted and filtered hydrol using RI
detector.
Table 3. 4: Physico-chemical characteristics of hydrol (starch molasses)
S.No
Description
i.
Dry Substance
70
ii.
Total Sugars
82
Ingredients
Glucose
56
Maltose
13
Maltotriose
Oligo Saccharides
10
Ash
1.56
pH
4.2
oligosaccharides, maltotriose, maltose and glucose as shown in Fig. 3.4.The Physico

Chemical characteristics of hydrol are shown in the Table 3.4
Since hydrol contained oligosaccharides, matotriose and maltose (Table 3.4), hydrol
was treated with Dextrozyme (Novozyme 2000) and used at dose of 0.5, 0.75, 1.0,
1.25, 1.5 ,1.75 and 2.0 units/g oligosaccharides, maltose and maltotriose at 60C (pH
4.5) for 24 h followed by fermentation up to 72 h in shake flask and fermenter studies.
It was found that 1 IU/g starch-based carbohydrate (properly diluted) was sufficient
41
for 100 % conversion to glucose and there was no reversion to maltose. Simultaneous
saccharification and fermentation studies were also performed. In these studies,
hydrol was treated with Dextrozyme (1 U/g starch-based carbohydrates) for 1 h at 60
C, followed by saccharification and fermentation at 40 C for addition 37 h.
Simultaneous saccharification and fermentation proved better with respect to glucose
productivity/h and was used in further experiments.
3.18
DETERMINATION OF GROWTH KINETIC PARAMETERS
At each harvest these parameters were recorded as before (Pirt 1975).Mass of dry
cells Cell mass (x, g cells/l) of S.cerevisiae, was found against dry cell ma and the
absorbance through Spectrophotometer. Coefficient of growth yield (Yx/s) found as g
cell mass/g substrate consumed. Volumetric rate of substrate consumption (Qs,g/l/h)
Ethanol production (QP, g/l.h or IU/l/h) calculated greatest value of the cell mass (g/l)
and product formation (g/l for ethanol and U/l for invertase versus time. The value of
(h-1) was found through the plot found with the help of ln x/x o against time;
Coefficient of product yield YP/X and YP/S were found by following formula:
YP/X = dp/dx and
YP/S = dp/ds
(3.1)
Product Formation (qp) and value of consumption of the substrate (qs)
were
determined by applying the equations:

qp = YP/X and
qs = / Yx/s
(3.2)
The volumetric rate of cell mass rate for the volume (Qx, g cells/l/h) also calculated
according to the need of the time and the maximum slope in plot of cell mass (g
cells/l) versus time
42
3.19 EFFECT OF TEMPERATURE

The effect of different temperatures (22, 24, 26, 28, 30, 37, 40, 43, 45, 47 and 50C)
was tested to find out the optimum temperature for the product formation by the S.
cerevisiae yeast cultures. Values of specific product yield of ethanol at different
temperatures in growth activation and deactivation phase were followed to calculate
thermodynamic parameters.
3.20 DETERMINATION OF THERMODYNAMIC PARAMETERS
3.20.1 Thermodynamics of cell mass and product formation
The energy of activation for cell mass formation (Ea(X)) and its energy of activation
were calculated through an Arrhenius was determined using Arrhenius model by
plotting natural log of specific growth rate (ln()) against the temperature (T) from
the following equation.
Ln( )= -Ea(X)/RT
where R (Gas constant) = 8.314 J mol-1 K-1
(3.3)
(3.4)
The doubling time of the cell mass was determined using equation
td = ln 2/
(3.5)
Activation energy for product formation was calculated by using the relationship:
ln(qpE) = -Ea(P)/RT
(3.6)
43
3.20.2 Thermodynamics of ethanol formation

Parameters for the thermodynamics of ethanol production were determined by
rearranging the Eyrings equation for that purpose and that is extended its value
according to Aiba 1973.
qP = T. kB/h.e
S*/R
e H*/RT
ln (qP /T) = ln (kB/h)+ S*/R - H*/RT
(3.7)
(3.8)
Where h is the Plancks constant and that values as = 6.63 10-34 J s and kB
(Boltzmann constant), [R/N] = 1.38 10-23 J K-1 where N (Avogadros number) = 6.02
1023 mol-1 and R (Gas constant) = 8.314 J mol-1 K-1.
For calculation of Gibbs free energy (G*) at each temperature, activation enthalpy
(H*) and activation entropy (S*) for ethanol/enzyme formation following equations
were used
G* (free energy of activation) = -RT ln(qP h/ kBT) (3.9)
H* (enthalpy of activation) = Ea(P)-RT
(3.10)
Where Ea(P) is the activation energy for product (ethanol,invertase) formation

calculated with the help of following equation
ln(qP) = -Ea(P)/RT
(4.11)
S* (entropy of inactivation) = (H*- G*)/T
(3.12)
while
44
3.21
EFFECT OF pH
The effect of initial pH of the medium ranging from pH 4.5, 5.0, 5.5 and 6.0 were
tested to find out the optimum pH of the medium for the formation of the product.
Then pH of the medium was and the pH of the medium was adjusted as describe on
dinner. The pH of the medium was adjusted as described in section 3.4.
45
CHAPTER 4
RESULTS AND DISCUSSIONS
4.1
MUTAGENESIS OF S. CEREVISIAE USING -RAYS
Gamma ray irradiation was done for S. cerevisiae. Different dosages of -rays were
used ranging from 0.2-1.4 kGy as described in materials and methods in order to
select a dosage rate at which 3 log kill could occur. The survival curve was prepared
which showed that the dosage rate of 1.0 kGy was optimal for 99.9 % kill.
For selection of mutant, two different approaches were used simultaneously. In first
strategy, the hyper ethanol producer mutant was selected by directly spreading the
irradiated cell suspension on agar plates containing 17 % sugars in blackstrap
molasses and 1.5 % DG along with the parental S. cerevisiae as control. We found out
that the native or parental culture was unable to grow under such conditions. In
second strategy preliminary selection was done through enrichment of the irradiated
cell suspension in liquid medium containing 17 % total sugars in blackstrap molasses
and 1.5 % DG before spreading on sucrose (12%)DG (1.5%) agar medium. The idea
behind the selection was based on the reaction of invertase product (glucose) with
glucose oxidase-peroxidase due to which pink zones would form in the presence of
ethanol produced or added in the selection plates. Larger and thicker pink zones
would display increased level of secretion of invertase. Using second strategy several
colonies (designated M1, M2, M3 to M10) showing bigger pink zones emerged on
agar medium containing 1.5 % DG and were selected as putative mutants. However
46
no mutant was obtained with the first approach. In order to check the mutants as
hyper ethanol and invertase producing mutant, further selection of mutant out of these
putative mutants was carried out on agar plates containing 12 % sucrose and 1.5 %
DG. These putative mutants were then propagated separately in liquid medium
containing 1.5 % DG and 12 % sucrose and spread separately on agar plates
containing above components. Out of 10, only one mutant (M9) was able to thrive
under these experimental conditions. In this way an ethanol hyper-secreting and
simultaneously thermotolerant mutant was obtained.
4.2
SUBSTRATE REGULATION OF INVERTASE AND ETHANOL

PRODUCTION
Carbon is the major constituent of all growing organisms and carbon sources play an
significant function in the production of enzyme production in a metabolic network.
Currently, there is an increasing awareness that the elemental composition of products
can be related to carbon source to support the formation of nucleic acids, amino acids
and the proteins they code. It has been confirmed that there exists a strong and
positive correlation between N/C values of genomes and proteomes of different
industrial organisms. Initially Table 1 shows that formation on a DGr and
thermotolerant mutant derivative S.cerevisiae M9 using other optimal operating
variables from literature (inoculum size 10 %, initial pH=5.5, and blackstrap molasses
medium described earlier) supplemented only with ammonium sulphate.
The mutant M 9 surprisingly showed maximum enzyme production stimulated by the
presence of the glucose, fructose and sucrose (components of blackstrap molasses).
Glucose confirmed that invertase production in DGr mutant was not related to
47
catabolite repression (Fig. 4.1, Table 4.1 & Table 4.2). Incorporation of sucrose has
been shown to increase invertase yield in many cases, but it must be emphasized that
the results reported in literature in this respect are not equivocal. In contrast to the
aforesaid investigation, mutant M 9 showed good invertase activity when the sucrose
and glucose were present in the media in the presence of glucose, which is in
accordance with studies reported by Rincon et al. (2001). Maximum cell mass yield
was regulated by 15-20% sugars. This suggested that increased production in the
mutant was correlated with cell mass formation. Enhanced cell mass formation by S.
cerevisiae M9 could lead to increased productivity of ethanol in large-scale
fermenters.
Preliminarily, extensive studies were undertaken to optimize Ffase and ethanol
production by varying process conditions like substrate type, pH of the medium,
carbon source concentration, and nitrogen additives. A conventional technique was
applied for changing a factor once by keeping the fermentation circumstances for
optimal production of Ffase and ethanol in 23-l fermenters. Time course studies of
invertase production by both wild and mutant organisms from glucose (representative
substrate) revealed that after 28 h, the wild organism supported only low level of
activity while the mutant organism supported maximum invertase synthesis.
Application of Luedeking and Pirt model using specific rate of invertase formation
indicated that production was connected with the growth and non growth and
invertase formation was not a purely primary metabolite.
However, a good
relationship existed between the enzyme titres and cell mass formation.
As mentioned earlier, the wild and mutant organisms were grown on 5 % glucose, and
5% glucose +10% sucrose media respectively and extra-cellular invertase was
48
checked on 10 % SDS-PAGE (Fig. 4.1). Invertase formation by glucose in the case of

wild organism was only marginal as mentioned earlier while the mutant strain showed
appreciable amount of invertase (Fig. 4.1, lane 1 and lane 4). When sucrose was also
present in the medium, glucose repressed synthesis of invertase in the wild organism
but its synthesis in the case of derepressed mutant was not significantly repressed by
glucose.,
Molasses is being mainly used in our country for production of ethanol. As mentioned
earlier, glucose and fructose in molasses cause catabolite repression on invertases
(Rincon et al. 2001). Different concentrations of sucrose, sucrose and glucose/
fructose combinations and molasses were in use to learn their consequences on
development and synthesis of extra-cellular and intracellular Ffase through wild its
mutated strain during the this research .The calculations of rates of reactions for Ffase
production by the wild and mutated cultures through 8 % and 10% sucrose (Fig.
4.2(a) and Fig 4.2(b) shoed that the action of the derived strain was giving more
values when fermentation time passes 24 hours. At 10% sucrose (Table 4. 1) the
system was saturated with respect to sucrose. The mutant was significantly improved
for both extracellular and intracellular Ffase production.
49
Fig 4.1:
Protein expression profile with and without sucrose in the growth
medium for both wild and derepressed mutant stains of Saccharomyces

cerevisiae.
Lane 1 &4= Protein expression profile on 5% glucose of derepressed mutant
of S.
cerevisiae; Lane 2-3 = 5% glucose of wild organism; Lane 5= 5% glucose + 10%

sucrose of wild organism; Lane 6= 5% glucose + 10% sucrose of derepressed mutated
organism;
Lane 7= Invertase standard (from yeast).
50
Fig. 4.2(a): Extracellular -fructo-furanosidase (Ffase) by parental cells (), and

mutant cells () and intracellular Ffase by parental () and mutant cells ()
following growth on 8 % sucrose in yeast medium which carries substrate.
51
Fig. 4.2 (b)

Fig. 4. 2 (b): Extracellular -fructo-furanosidase (Ffase) by parental cells ()
and mutant cells () and intracellular Ffase by parental () and mutant cells
() following growth on 10% sucrose in yeast medium which carries substrate.
52
An other representative figure (Fig.4.3) indicating concomitant production of ethanol,

extracellular and intracellular Ffrase production by M9 mutant revealed that ethanol
made of the parental and mutant (Table 4.1) This indicated that Ffase formation was
dependent on of both organisms.
Fig. 4.3: Kinetics of production of ethanol (), cell mass () and substrate in the
medium ()extracellular (inverted open triangle) and intracellular () following
growth of mutant cells body.
53
Table 4.1: Comparative fermentation kinetic parameters of S. cerevisiae and its

mutant derivative M9 for extra-cellular and intracellular Ffase production and
specific growth rate on different concentrations of sucrose in 23 l fermentor
(working volume 15 l) at 30 C
C. source
QP (IUl-1h-1)
Yp/S (IU g-1)
Ypp/x (IUg-1 cell)
(h-1)
Extracellular Ffase by wild cells

Suc 8 %
650
67
1130
0.21
Suc 10 %
668
73
1133
0.19
Suc 12 %
678
75
1135
0.15
Extracellular Ffase by mutant cells

Suc 8 %
750
128
1677
0.23
Suc 10 %
775
132
1556
0.21
Suc 12 %
980
136
1690
0.17
Intracellular Ffase by wild cells

Suc 8 %
750
261
1911
0.21
Suc 10 %
754
265
2377
0.19
Suc 12 %
765
268
2378
0.15
Intracellular Ffase by mutant cells

Suc 8 %
1050
391
2867
0.21
Suc 10 %
1052
400
3566
0.19
Suc 12 %
1053
402
3567
0.15
Each value is a mean of n=2 replicates. Standard error between replicates varied
between 4-5% of mean values and has not been presented. Suc=sucrose.
54
Table 4.2: Comparative fermentation parameters of S. cerevisiae and its mutant

derivative M9 for production of extracellular and intracellular Ffase production
on different substrates in 23 l fermenter at 30 C
C. source QP (IUl-1h-1)
Yp/S (IU g-1)
Ypp/x (IUg-1 cell)
(h-1)
Extracellular FFase by wild cells

Suc.+Glu
510
53
889
0.20
Suc.+Fru
526
53
890
0.20
Molasses
535
53
890
0.20
Extracellular FFase by mutant cells

Suc.+Glu
1030
101
1677
0.21
Suc.+Fru
1043
98
1556
0.21
Molasses
1045
105
1690
0.21
Intracellular FFase by wild cells

Sucrose
3458
463
4111
0.20
Suc.+Glu
650
189
1911
0.20
Suc.+Fru
654
143
2377
0.20
Molasses
665
192
1932
0.20
Intracellular FFase by mutant cells

Suc.+Glu
975
375
3836
0.21
Suc.+Fru
980
400
3900
0.21
Molasses
982
402
3950
0.21
These are average readings of the data that covers the time and bears one through
different two .The target were. Standard error between replicates varied between
4-5% of mean values and has not been presented. Suc=sucrose Glu=glucose and Fru=
Fructose.
55
Molasses used by most distilleries has 15% total carbohydrates for fermentation for
24 h. To simulate commercial ethanol production process, all studies were performed
using 15% total sugars as present in molasses ( sucrose 10, glucose and fructose 5 At
5% Glucose as Carbon sources it supported 50 IU per l per h productivity in the wild
cells; the mutant was 5-fold improved for extracellular enzyme formation as described
earlier Ricon et al. (2001). It was also concluded this improved mutated strain gives a
rise up to 2-and 1.5-fold with respect to synthesizing extra-cellular and intracellular
Ffase respectively. Ali and Haq (2007) isolated a derepressed mutant after mutagenic
treatment with ethyl methane sulfonate. Maximum extracellular Ffase yield of
3.461.1 IU/g sucrose was exhibited by the best mutant and was almost 100-fold less
active than mutant M-9 reported in this study. Ul-Haq et al. (2008) isolated a multiple
mutant with sequential mutation of wild and newly developed mutant cells of S.
cerevisiae in step by step mutation with UV, MNNG, and EMS and optimized
fermentation time 48 h, sucrose concentration 5.0g/l, initial pH 6.0 and inoculum size
2.0% (v/V) and reported enzyme yield was made upto 2.1 IU/g sucrose when starting
pH was recoded as 6 and sucrose concentration was 5.0 gl-1.
Addition of glucose and fructose, repressed the wild cells to support 34% less enzyme
synthesis (Table 4.2). Usually-sources when supplied hurriedly for the catabolic
pathway for proteins formation in the reactors. (Bohm and Boos 2004, (Bohm and
Boos 2004; deGroot et al. 2003) to synthesize more enzyme. That was also conclude
by the fresh research that it gives an improved rate of production etc.(Ashokkumar
and Gunasekaran 2002; Montiel-Gonzalez et al. 2002; Yanai et al. 2001).
The yeast strain was tested and optimized in the fermenter of 23 liters capacity
(working volume 15 liter) and the optimized conditions were used to perform the
56
experiments using the fermenter of 150 liters capacity. The mutant strain proved
more productive with respective to ethanol (7.5 % w/v, 95.42 % of the theoretical
yield) and 9.4 g/l cell mass and consumed almost all sugars (98.6% w/v) at 43 oC and
found alike to one presented earlier (Rajoka et al. 2005) for 28 h fermentation.
Whereas Banat et al. (1995) utilized 99.6% sugars from 25-50 % total sugars in 27 h.
The strain showed improved behaviour, for the cell mass production, substrate
consumption and ethanol production (Tables 4.3 to 4.14) with some negligible
decrease at the fermentations at higher working volume (150 liters). It was observed
that the fermentation at this scale, digitally controlled through a microprocessor
computer programming, is easy to operate and control as compared to the shake flask
fermentation.
4.3
INITIAL OBSERVATIONS
Time course study was carried out at 15 liter fermenter for 28 h to observe ethanol
production, substrate consumption and cell mass and is shown in the figures 4.4 to
4.12 and the tables 4.3 to 4.17). At the start of the fermentation for 4 h, the cell mass
showed the lag phase and then the log phase started and by reaching at the peak value
of 9.0 g/l, the system kept a steady state condition up to 28 h.
4.4
SUBSTRATE CONCENTRATION DEPENDENT FORMATION OF

ETHANOL
Further studies on regulation and maximization of ethanol biosynthesis in a

thermotolerant mutant derivative of S. cerevisiae has been investigated using two
substrates (blackstrap molasses and starch molasses with different concentrations),
nitrogen sources, inoculum sizes, pH of the medium, temperature, in batch
57
fermentation in a 23 liter fermenter. The production of intracellular invertase was

improved 1.5 times following media optimization involving one-at-a-time approach in
this fermenter. Ddependent variables studied were cell mass, ethanol titer.
Fig. 4.4(a): Effect of sugar concentrations [ 5 %(( , ), 8 %( , ) 10 %(,),

12 %(,) and 15 %(
,)] in molasses on ethanol production by native (empty
symbols), and mutant (filled thick symbols) cells of S. cerevisiae.
58
Fig.4.4 (b): Effect of sugar concentrations [ 5 %(, ), 8 %(, ) 10 %(,), 12

%(,) and 15 %(
,)] in molasses on cell mass production by native (empty
symbols), and mutant (filled thick symbols) cells of S. cerevisiae.
Fig. 4.4: Effect of sugar concentrations [ 5 %(, ), 8 %( , )
10 %(,), 12
%(,) and 15 %(, )] in molasses on substrate consumption by native empty

symbols), and mutant (filled thick symbols)) cells of S. Cerevisia.
59
Table 4.3: Kinetics parameters for cell mass production substrate consumption
and ethanol formation by the native (N) and mutant (M) cells of S. Cerevisiae at
different concentrations of sugars.
Concentratio
n
5%
N
M
8%
N
M
12 %
N
M
15 %
N
M
17.5%
N
M
20 %
N
M
Qx
g
cell/
l/ h.
Qs
g/l/h
0.32
0.36
0.23
0.28
5.35
6.04
0.33
0.35
0.26
0.30
0.33
0.35
Qp
g/l/h
X/S
P/X
P/S
qp
g/g/h
qs
g/g/h
g/g
g/g
g/g
1.35
1.44
0.08
0.12
5.6
7.1
0.47
0.51
1.05
1.13
3.85
4.04
7.35
8.57
1.56
1.70
0.09
0.13
6.25
7.67
0.45
0.49
1.21
1.24
4.12
4.4
0.29
0.32
7.39
8.8
1.86
2.00
0.12
0.14
6.45
7.87
0.44
0.45
1.41
1.43
4.42
4.80
0.32
0.34
0.35
0.41
8.5
9.0
2.5
2.8
0.14
0.16
6.85
7.89
0.47
0.50
1.25
1.50
4.65
5.10
0.31
0.32
0.32
0.43
7.5
7.9
2.3
3.1
0.12
0.12
7.0
7.78
0.45
0.50
2.33
2.53
4.44
4.96
0.25
0.28
0.31
0.45
6.5
7.9
2.1
2.9
0.08
0.11
6.23
7.15
0.42
0.46
1.63
2.00
3.56
4.56
F value for substrate concentration for QP=59.2 with p = 0.0000 at p 0.05

F value for organisms for QP =40.68 with p = 0.0000 at p 0.05
F value for substrate concentration x organisms for QP =5.29 with p = 0.002 at p
0.05.
Ethanol yield, ethanol specific and volumetric rate of formation, invertase titer, and
related kinetic parameters. It was confirmed that ethanol production in the derepressed
and thermotolerant mutant is sufficiently uncoupled from catabolite repression and
defolding of proteins of the metabolic pathway.
Substrate concentration dependent formation of ethanol studies (Table 4.3) revealed
that 15, 17.5 and 20% total sugars were not significantly different with respect to
60
supporting formation of ethanol rate (f value = 59.2, p = 0.000 at p 0.05) in the

mutant derivative. It was, however, interesting that both native and the mutant
organisms were not statistically different in supporting ethanol productivity on 15%
total sugars in molasses (Table 4.3). However, lower % of the sugars gives lower
effect on the cell mass and ultimately on the ethanol production. Stenberg et al. (2000)
have proved that sugar levels of more than 15 % can inhibit the cell activity and
ultimately reduces the overall production of alcohol. Shigeru et al. (1997) have
utilized 25 % total sugars from molasses at 35 oC and 9.1% ethanol was produced in
34 h fermentation time. Kim et al. (2006) used 20% glucose at the temperature of 3040 oC for 24 h. Najafpour et al. (2004) used 25, 35 and 50% of total sugars and
produced 0.29 g yield of ethanol.
Lakhana et al. (2004) compared the competence of manufacture of Ethanol was
recorded as good in the previous researchers that experiment was not fuly regularize
and automatized. Results of productivity by the mutant derivative in current studies
are comparable to the best fermentation process studied by these authors.
61
Table 4.4: Time dependent molasses concentration on ethanol and cell mass
Formation
Time
(h)
0
4
8
12
16
20
24
28
Sts
5%
N
M
N
M
N
M
N
M
N
M
N
M
N
M
N
M
8%
10 %
12 %
15 %
X
(g/l)
S
(g/l)
P
(g/l)
X
(g/l)
S
(g/l)
P
(g/l)
X
(g/l)
S
(g/l)
P
(g/l)
X
(g/l)
S
(g/l)
P
(g/l)
X
(g/l)
S
(g/l)
P
(g/l)
0
0
0.1
0.4
0.4
0.8
0.5
1
0.8
1.7
1.3
3.3
2.0
3.3
2.8
4.2
50
50
47
43
45
40
38
32
31
24
27
21
23
17
20
13
0
0
0
3
3
5
6
7
11
18
16
27
17
34
17
34
0
0
0.3
0.5
0.7
1
1.5
1.4
2
2.7
3.4
3.6
4.0
3.9
5.3
6.6
80
80
71
43
45
40
38
32
31
24
27
21
23
17
20
13
0
0
1
2
6
7
6
9
19
21
33
35
19
41
19
41
0
0
0.5
0.8
0.9
1.4
1.9
2.4
3.7
5.6
4.9
7
6.0
7.9
6.7
8.3
100
100
87
79
73
66
65
51
53
45
41
33
32
21
23
15
0
0
7
15
9
22
11
30
18
32
24
36
34
43
34
43
0
0
0.6
0.8
1
1.9
2.5
2.9
4.1
5.8
4.7
7.1
5.4
7.7
6.8
7.4
120
120
112
109
96
81
72
65
51
47
39
29
26
17
14
11
0
0
1
2
6
7
6
14
19
34
37
45
50
70
50
71
0
0
0.6
0.8
1
1.9
2.5
2.9
4.1
5.8
4.7
7.1
5.4
7.7
6.8
7.4
150
150
130
119
97
81
68
57
51
43
34
23
20
15
11
4
0
0
2
5
6
10
11
19
28
42
51
57
61
74
61
74
Table 4.5: Substrate concentration dependent kinetics parameters for ethanol

and cell mass formation and substrate utilization by the mutant organism
calculated using data in Table 4.4
Concentration
Qx
Qs
Qp
Y X/S
Y P/X
Y P/S
qp
qs
5%
0.16
0.28
3.04
2.04
0.05
7.1
0.34
1.13
4.04
8%
0.20
0.30
3.57
1.70
0.05
7.67
0.29
1.24
4.4
12 %
0.27
0.32
4.8
2.0
0.12
7.87
0.35
1.43
4.8
15 %
0.34
0.4
5.1
2.8
0.26
7.89
0.39
1.5
5.1
62
Fig. 4.5(a):
Effect of sugar concentration on the production of ethanol by the
wild (open symbols) and mutant derivative of S. cerevisiae in 15 liter working

volume fermenter.
Fig. 4.5(b):
Effect of sugar concentration on the cell mass production by the
wild (open symbols) and mutant derivative of S. cerevisiae in 15 liter working

volume fermenter.
63
Fig.4.5(c):
Effect
of
sugar
concentration
consumption by the wild (open symbols) and
on
the
Substrate
mutant derivative of S.
cerevisiae in 15 liter working volume fermenter.
4.5
EFFECT OF SUBSTRATE SOURCES
Molasses and Hydrol were used as the carbon sources to see their effects on both
strains for ethanol fermentation. Since hydrol contained oligosaccharides, matotriose
and maltose (Table 3.3), hydrol was treated with Dextrozyme (Novozyme 2000) and
used at dose of 0.5, 0.75, 1.0, 1.25, 1.5 ,1.75 and 2.0 units/g oligosaccharides, maltose
and maltotriose at 60C (pH 4.5) for up to 24 h (called saccharification) followed by
fermentation for 28 h or hydrol was treated continuously at 60 C, after fermentation
upon 40 C for up to 72 h (simultaneous saccharification and fermentation, Rashad
64
2003). Simultaneous saccahrification and fermentation proved better and was used in
these experiments. The data in Tables 4.6 and 4.7 show the comparison of these two
carbon sources. Molasses has got better effects on fermentation of ethanol. Molasses
gave 74 g/l and Hydrol gave 60 g/l of ethanol. For the substrate consumption also,
molasses proved better and compares favorably well with the research presented by
Gough et al. (2001). Banat et al. (1996) used whey permeate as carbon source at 45
o
C and reported cell mass 0.9 g/l to produce 43 g/l of ethanol. Win et al. (1996) used
glucose and molasses as the carbon sources, which resulted in 0.23 g yield of ethanol
with the mesophilic range of fermentation temperatures.
65
Table 4.6: Effect of different substrate sources on ethanol and cell mass
formation with time dependent substrate consumption from both sources
Time
(h)
12
16
20
24
28
Strain
N=Native
M=Mutant
Molasses
Hydrol
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
=0.32
=0.28
150
150
150
150
0.9
139
0.87
142
1.1
138
0.98
140
3.1
106
13
2.6
108
11
4.2
104
20
3.5
106
17
4.2
70
15
3.87
73
11
68
23
4.8
71
20
5.4
37
28
5.
40
23
5.6
34
41
5.2
38
39
6.9
15
51
5.7
16
34
9.1
12
55
5.9
14
51
57
7.5
50
9.5
74
8.2
58
57
7.5
50
9.5
74
8.2
58
66
Table 4.7: Kinetics for mutant strain for substrate consumption and product
formation parameters from molasses and hydrol
Qx
Qs
Qp
YX/S
YP/X
YP/S
qp
qs
Molasses
0.32
0.38
6.16
3.12
0.064
7.89
0.506
3.12
5.0
Hydrol
0.28
0.34
6.08
2.14
0.055
6.10
0.35
1.70
6.4
Carbon
Sources
Fig. 4.6(a): Effect of substrate sources on cell by both organisms, wild and
mutated.
67
Fig.4.6 (b): Effect of substrate sources on substrate consumption by wild and

mutated organisms.
4.6
REGULATION OF ETHANOL PRODUCTION BY NITROGEN

SOURCES
These, together with inorganic and organic take part significantly for the regulating of
production of enzymes of metabolic network of a particular product under study.
Inorganic nitrogen sources are consumed quickly and normally cause repression of
enzyme synthesis due to formation of ammonium repressible entity a protein (Bohm
and Boos 2004) whilst it is required for the micro-organisms growth and proteins
formation. Hence both the sources were used to observe their regulatory role in
ethanol fermentation in 15 liter working volume fermenter. The dependence of
ethanol production on nitrogen sources is presented in Table 4.8. The influence of all
nitrogen sources on all fermentation attributes (kinetic parameters) of ethanol
68
production was quite significant (Table 4.9). Ammonium sulphate was optimal
nitrogen source for up-regulation of intracellular enzyme synthesis of metabolic
network (glycolytic pathway) of ethanol. The yields on media containing urea, or corn
steep liquor were lower than those with ammonium sulphate.
Time course study of the fermenter to see the effects of nitrogen sources (0.246%
nitrogen) on the mutated strain of S. cerevisiae for the ethanol production, cell mass
and substrate consumption is shown in figures (4.7a - 4.7c). Among these three
ammonium sulfate proved more useful regarding all values for ethanol and cell mass
synthesis with substrate consumption (Table 4.8 & Table 4.9) and showed 75 g/l
ethanol production, 9.4 g/l cell mass and 148 g/l the substrate consumption where as
the earlier reports showed lesser values (Bailey, 1991; Rajoka, et al. 2005).
69
Table 4.8: Time dependent effect of different nitrogen sources on ethanol, cell
mass and substrate present in 15 liter working volume fermenter
Time
Strain
(NH4)2SO4
Urea
X
h
12
16
20
24
28
N = Native
(g/l)
M = Mutant = 0.30 (g/l) (g/l)
X
(g/l)
= 0.23
CSL
(g/l) (g/l)
X
(g/l)
= 0.30
(g/l) (g/l)
15
150
150
150
150
150
1.5
138
1.1
139
0.9
139
1.7
136
1.2
139
1.2
137
2.1
106
2.1
109
3.1
112
2.3
104
32
2.3
107
3.2
110
3.7
67
11
4.2
69
3.8
71
65
46
4.2
67
14
4.1
68
17
5.2
33
28
5.2
35
19
5.
39
16
5.4
31
62
5.4
33
34
5.2
36
29
6.4
11
56
6.5
12
37
5.9
14
31
7.7
75
6.7
11
45
6.1
12
38
7.8
61
7.6
50
7.4
31
8.4
69
7.8
61
7.8
47
7.9
63
7.8
52
6.7
31
9.4
71
66
8.2
47
70
Table 4.9: Kinetics of product formation of ethanol and substrate consumption

parameters for mutant strain of S. cerevisiae using N-sources
N-Sources
Qx
Qs
Qp
Y X/S
Y P/X
Y P/S
qp
qs
(NH4)2SO4
0.30a
0.335a
7.05a
3.75a
0.047c
11.19a
0.53a
3.35a
6.38a
Urea
0.23b
0.325c
6.16b
2.88b
0.054a
8.62b
0.47b
1.98b
4.26c
CSL
0.30a
0.329b
6.12c
1.96c
0.053b
5.95c
0.42c
1.78a
5.66b
Fig.4.7 (a): Ethanol production from 30 C by both wild (open symbols) and
mutant (closed symbols) strains in 23 liter working volume fermenter. All
conditions were kept constant except nitrogen sources were altered and
maintained at a concentration of 0.246 % nitrogen.
71
Figure 4.7(b): Ethanol production from 30 C by both wild (open symbols) and
maintained at a concentration of 0.246 % nitrogen
72
Figure 4.7: Ethanol production from 30 C by both wild (open symbols) and
maintained at a concentration of 0.246 % nitrogen
73
4.7
EFFECTIVENESS OF AIR ON PRODUCTIVITY OF ETHANOL
Flow of air was maintained at starting flow rate of ethanol. In these experiments all
the parameters were kept in touching with slow and then fast rate of proceedings.
Details are given in the Table 4.10.
Table 4.10:
Effectiveness of air flow on ethanol and cell mass production
during time course uptake of sugars from molasses
Time
Strain
N=Native
M=Mutant
0.1
vvm
X
(g/l)
(h)
0
4
8
12
16
20
24
28
N
M
N
M
N
M
N
M
N
M
N
M
N
M
N
M
0
0
0.2
0.78
2
3.4
2.8
3.5
3
4.2
4.3
4.9
5.8
6.6
5.8
6.6
0.2
vvm
(g/l) (g/l)
150
150
130
140
118
113
97
68
43
25
19
12
13
8
9
5
0
0
4
7
13
18
24
29
45
53
56
58
61
65
61
64
X
(g/l)
0
0
0.2
0.84
1.6
3.8
3
3.9
3.7
4.7
4.8
5.9
7.0
7.4
7.0
7.4
0.3
vvm
(g/l) (g/l)
150
150
127
132
111
102
90
62
35
21
17
10
11
7
7
2
0
0
8
12
12
30
26
34
48
59
64
67
67
70
67
69
X
(g/l)
0
0
0.1
0.7
1.2
3
3
4.3
3.3
4.2
4.1
6
6.4
7
6.6
6.4
0.4
vvm
(g/l) (g/l)
150
150
129
137
117
105
92
63
42
28
22
17
16
10
11
7
0
0
2
4
5
11
20
26
27
37
32
43
48
60
48
60
X
(g/l)
0
0
0.1
0.8
1
2.6
3
4.4
2.8
3.7
3.3
4.8
5.0
6.1
6.1
6.4
(g/l) (g/l)
150
150
132
139
119
109
98
67
46
28
26
23
20
11
15
7
0
0
3
4
7
7
12
15
11
34
23
39
37
42
37
42
74
Table 4.11: Airflow rate dependent kinetic parameters for ethanol formation and
substrate consumption by the mutant strain by maintaining all other process
variables constant except airflow rate, which had different values.
Air flow rate
Qx
Qs
Qp
Y X/S
Y P/X
Y P/S
qP
qS
0.1 v/vm
0.23
0.23
5.12
2.29
0.045
9.69
0.44
2.22
5.11
0.2 v/vm
0.34
0.26
5.28
2.46
0.05
9.22
0.46
3.16
6.8
0.3 v/vm
0.19
0.23
5.08
2.47
0.04
9.38
0.41
1.77
3.8
0.4 v/vm
0.17
0.23
5.10
2.46
0.039
5.56
0.29
1.58
3.7
Initially air flow rate was maintained at 1 vvm for 8 h to get more cell mass. Keeping
all optimized parameters, air flow rate was altered for the purpose of learning the
outcome on ethanol and cell mass formation in time course study.
75
Fig. 4.8(a): Ethanol by both Native and mutated strains of S. cerevisiae at 30 C

All other process variables were kept constant except air flow rate, which was
changed from 0.1 to 0.4 vvm.
76
Fig.4.8(b): Cell growth by both Native and mutated strains of S.cerevisiae at 30

C all other process variables were kept constant except air flow rate, which was
changed from 0.1 to 0.4 vvm.
77
Fig: 4.8: Substrate Consumption by both Native and mutated strains of S.

cerevisiae at 30 C All other process variables were kept constant except air flow
rate, which was changed from 0.1 to 0.4 vvm.
Vitolo (2003) allowed growing Saccharomyces cerevisiae on blackstrap molasses

with out aeration and at different pH of the medium (4.0, 4.5, 5.0, 5.5, 6.0, & 6.5).
The highest production of ethanol (8.5 g l-1 h-1) and invertase (238 Ul-1) were attained
at 4.5 and 5.0 and 5.5, respectively. The invertase formation would be subjected to
carbohydrate repression/depression mechanism and to the culture conditions
employed, such as pH by them.
78
4.8
EFFECT OF AGITATION
At the larger scale like pilot size it is vital to supply Oxygen with the help of a
mechanical set up known as agitator to get the air compressed. Further studies are
required to know the effect of oxygen on the material present in an anaerobic The
rate of oxygen at which it is being transferred and the agitational intensity is directly
proportional to each other.
In the process of fermentation, oxygen transfer is possible through the bubble as the
liquid transfers in a gas. Ultimately the gas is shifted to the microorganism. This
research for the oxygen shifting from a liquid to gas and gas to liquid is very
necessary and it is done by air flow rate and agitation. They have a pivotal role for
mass transfer of cell mass, substrate, product and temperature. Various rates of
agitation were observed in the reactor from 200 to 400 rpm but the most effective was
the rate of 300 rpm (7.4 % ethanol production, 97 % sugar utilization and 9.6 g/l cell
mass production).Where as Oniscu et al (2002) observed the rates at higher values
upto 700 rpm when it was experienced for the bioreactors of smaller volumes of 5
liters.
Ahmad et al (1994) has established a relationship of oxygen transfer and the rate of
agitational intensity in a bioreactor during the process of fermentation .He has proved
that when an the agitational intensity of 300 to 600 will give a rise to oxygen transfer
rate from 8.94 to 38.63 mmol l-1h-1 .He has further found that of the rate of transfer of
air is enhanced from 0.21 l min-1 to 1.05 l min-1, the oxygen transfer rate increased
from 5.7 mmoll-1h-1 to 20.5 mmol l-1h-1. This is done in all sorts of fermentation
processes that when a rate of gas transfer occurs, the oxygen transfer increases this is
very classical, higher productive and supportive one.
79
Table 4.12: Effectiveness of agitation for ethanol productivity and for the growth
of cells during the time dependent substrate uptake standard working
conditions.
Time
Strain
12
16
20
24
28
300 rpm
(g/l)
(g/l)
15
150
150
0.8
143
1.6
139
3.6
400 rpm
(g/l)
(g/l)
(g/l)
150
150
150
139
0.8
140
1.2
131
1.5
137
126
3.5
117
3.3
123
22
4.4
118
3.9
107
3.6
118
32
87
3.9
72
16
3.9
79
27
4.8
74
4.7
67
19
4.6
73
34
5.
39
11
5.
33
39
4.6
38
31
5.9
28
13
5.6
21
34
5.8
28
36
5.9
18
23
5.8
14
47
5.9
21
34
6.8
11
28
6.9
45
6.8
15
36
5.9
35
7.2
11
60
13
61
6.6
41
9.6
74
8.3
67
5.9
35
7.2
11
60
13
61
6.6
41
9.6
74
8.3
67
(h)
200 rpm
(g/l) (g/l) (g/l) (g/l)
80
Table 4.13: Agitation dependent kinetics parameters of mutant strain for

substrate consumption, and product formation in 15 liter (working volume)
fermenter under standard working conditions.
Agitational
Qx
Qs
Qp
YX/S
200 rpm
0.22
0.27
6.04
1.7
0.04
300 rpm
0.29
0.40
6.12
2.54
400 rpm
0.24
0.34
6.00
450 rpm
0.20
0.30
500 rpm
0.17
0.24
YP/X
YP/S
qp
qs
6.21
0.28
1.37
5.5
0.06
7.71
0.50
2.2
4.8
2.79
0.057
8.3
0.46
1.2
4.2
4.7
2.16
0.05
6.58
0.35
1.22
4.1
1.22
0.037
5.97
0.31
1.08
3.6
intensity
The data given in the table is an average of the tow readings .The errors between
values were small there fore it is not shown in the data.
Fig.4.9(a): Effectiveness of agitation for ethanol productivity from 15 % total

sugars from both of the organism. The data given in the table is an average of the
two readings.
81
Fig.4.9 (b): Effect of agitation (rpm) on cell mass formation from 15% total
sugars. The data given in the table is an average of the tow readings.
82
Figure 4.9(c): Effect of agitation on substrate consumption from 15% total

sugars in molasses from the organism. The data given in the table is an average
of the tow readings .The errors between values were small there fore it is not
shown in the data
4.9
EFFECT OF ADDITIVES
Tween 80 and NaF were used as additives to enhance production of ethanol but the
data obtained (Table 4.14 and 4.15) show that these additives had no effect on the
production of ethanol within 24 h of fermentation. The data shown in the previous
results is very quite matching with the data presented by Patel et al. (1998) who
showed that the addition of tamarind supplement can only enhance the production
after 72 h of reaction time, however, no enhancement was observed with in 24 h.
83
Table 4.14:
Effect of different additives on the ethanol and cell growth, time
dependent consumption of 15 % total sugars in molasses (pH =5.5) under

optimized working conditions
Strain
Tween 85
Time
(h)
0
4
8
12
16
20
24
28
NaF
N=Native
M=Mutant
N
M
N
M
N
M
N
M
N
M
N
M
N
M
N
M
X
(g/l)
=0.19
0
0
0.9
1.8
3.1
3.7
3.6
4.9
5.
6.2
5.9
7.1
6.3
7.2
6.3
7.2
S
(g/l)
P
(g/l)
150
150
143
145
134
137
67
62
45
36
15
12
8
5
8
5
0
0
12
15
21
28
34
42
38
44
38
49
26
56
26
68
X
(g/l)
=0.22
0
0
0.7
1.
2.4
2.8
3.
3.7
4.8
5.7
5.
6.1
5.6
7.3
5.6
7.3
S
(g/l)
150
150
140
137
123
115
54
48
40
32
26
21
13
09
13
09
P
(g/l)
0
0
4
9
16
11
12
24
13
36
23
47
29
52
29
52
84
Fig.4.10 (a): Effectiveness of additives shown for ethanol
productivity from
molasses (total sugars 15 %, pH =5.5) under optimized working conditions.
Figure 4.10(b): Effect of additives on cell mass synthesis from molasses

sugars 15 %, pH =5.5) under optimized working
(total
conditions. Additives were
Tween 80 for parental () and mutated () strain respectively.
85
Fig.4.10: Effect of additives on substrate consumption by the native (open

symbols) and mutant strain (closed symbols) of S. cerevisiae.
4.10
PRODUCTION FROM HYDROL
The generally the attractive characteristic of this recently improved microorganism is

its steadiness or firmness during its genetic improvements for the further
productivity.(Rincon et al. 2001). Effect of substrate concentration on kinetic
parameters (Fig.4.11) revealed that the sugar concentration of 15% gave highest
values of all kinetic parameters except QP, which was maximum on 20 % TRS in
hydrol. It was observed that high sugar was accompanied by increased glycerol (1.5
g/l), citric acid (0.5 g/l) and acetic acid (0.15 g/l) production. Low sugar concentration
ultimately resulted in increase of ethanol production in the medium.
86
Fig.4.11: Effect of substrate concentration on specific growth rate (), specific

substrate consumption (qS), volumetric rate of product formation (QP), product
yield (YP/S) and specific productivity (qP) in 23-l fermenter using Dextrozymepretreated hydrol as substrate, and corn steep liquor (25 g/l) as nitrogen source.
Initial flow rate was 1 vvm for 8 h followed by 0.25 vvm in agitated vessel (250
rpm) at 30 C.
Therefore optimized sugar concentration necessary for ethanol production was 15%
(W/V) and gave 98 % theoretical yield of ethanol. To avoid the inhibitory effects of
substrate and ethanol, 15% total reducing sugars (TRS) in hydrol were used in further
optimization studies. Studies on Dextrozyme pretreatment of hydrol in shake flask
and fermentor studies concluded that one IU Dextrozyme/g tri-saccharides and
oligosaccharides was sufficient to get 100 % glucose and produce ethanol. Among
nitrogen sources (ammonium sulfate, urea and corn steep liquor), corn steep liquor
87
(25 g/l) supported maximum ethanol production (74 g/l) from hydrol and was used as
only additive to hydrol for ethanol production. It was observed that with the selected
mutant derivative maximum values of ethanol (49 g/l) were obtained with 1 vvm
aeration rate for rest 28 h at 40 C at 250 rpm agitation speed in 23-l fermenter,
following growth on 200 g TRS/l.
To assess the mutational effect on thermostability of the genetic make up of the DG r
and thermotolerant mutant organism, the inoculated hydrol fermentation medium was
incubated at 20,22, 24, 26,28, 30,35,40, 43, 45 and 47C (Fig. 4.12) in 23 liter
fermenter (see Materials and Methods), keeping all other fermentation conditions
constant.
Ribosome become slowly when the temperature reaches at lower values and at higher
values of the temperature the cell membrane does not allow the feed nutrients in the
microorganism. There fore the affinity and the requirement of the energy is enhanced
in the cells (Aiba et al. 1973) for ethanol production by the organisms. Average
specific ethanol productivity was enhanced when temperature was increased from
15-35C. Maximum ethanol specific productivity (2.1 g/g dry cells /h) was supported.
But when there is an involvement of wild organism, this temperature was 30-35 C.
This temperature range is the normal range reported for other wild strains of S.
cerevisiae (Rajoka et al. 2005). Conversely, specific productivity decreased over
35C, although maximal growth occurred at 35C. Reduced ethanol productivity was
probably because of the metabolic networks inactivity, when the temperature range is
higher than the optimum.
The capability of fermentation of that of the microorganism was exaggerated by the
temperature of fermentation and was measured as product yield, volumetric
88
productivity and specific productivity. The values of above attributes increased up to

40 C (Table 4.15). A major difference was recorded and the data fond as significant.
The difference among the specific rate of ethanol production (qp), ethanol yield,
specific ethanol yield and volumetric rate of ethanol with temperature (Table 4.15), as
was its effect on specific growth rate (), cell mass formation rate, substrate
consumption rate and cell yield (Table 4.16). Thus effect of temperature on all kinetic
parameters was evident from the high values of F (Table 4.15 and Table 4.16), which
signified that influence of temperature at 40 C was found optimum for ethanol
production by the deoxyglucose and thermo-tolerant mutant derivative of S.
cerevisiae.
Table 4.15: Kinetic parameters of S. cerevisiae M -9 for ethanol production
following growth on 15 % sugars in Dextrozyme-pretreated hydrol in a fully
controlled 23-l fermenter
Temperature QP (g/ lh)

YP/S (g/ g substrate)
C
20
1.3g
0.27e
4.30c
25
1.6f
0.31d
6.70ab
YP/X (g/ g cells) qP (g/g h)
30
2.2d
0.47b
7.80ab
1.69b
35
2.6b
0.48b
8.00ab
2.08a
40
2.8a
0.50a
8.25a
2.10
45
2.5c
0.40c
7.10ab
1.49c
47
1.8e
0.32d
6.50b
0.62d
50
0.9h
0.15f
3.7c
0.41e
356.1
239.4
10.0
356.1
0.0000
0.0000
0.0002
0.0000
0.66d
1.47c
________________________________________________
89
An average was presented from the of two experiments. An error amongst 3.5 to 5.0
%. Various alphabetical letters show the significance at p0.05.
Table4.16: Temperature effect on biomass formation and substrate consumption

by
S. cerevisiae M-9 during ethanol production following growth on 15 %
sugars in Dextrozyme-pretreated hydrol in a fully controlled 23-l fermenter.

______________________________________________________
Temperature QX (g/ lh)
YX/S (g/g)
QS (g/lh)
(1/h)
C
20
0.29e
0.071bc
2.6g
0.14e
25
0.46c
0.080ab
4.0e
0.20d
30
0.56b
0.084ab
4.7c
0.21c
35
0.60a
0.088a
4.8b
0.24b
40
0.61a
0.091a
5.0a
0.26a
45
0.46c
0.078a
4.3d
0.21c
47
0.43d
0.056c
3.9f
0.19d
50
0.24f
0.035d
2.1h
0.11f
257.4
276.9
290.8
140.02
0.000
0.0000
0.000
0.000
_________________________________________________________
An average was presented from the two experiments. An error amongst 3.5 to 5.0 %.
Various alphabetical letters show the significance at p0.05.F and P values indicated
that the temperature effects noteworthy.
90
Fig. 4.12: Determination of activation energy for growth (a) and formation of
Ethanol with the help of native and the mutated strain. using hydrol based
medium (dextrozyme treated 15% sugars containing hydrol) using Arrhenius
relationship.
91
Highest values for product, cell mass growth and death rate fo the cells was observed
as increasing upto the range of 40 C in the case of mutant derivative. Experiment
showed that the temperature in dependent of these parameters presented in the table
Figure 4.12(a) and
Figure 4.12(b). The calculated data for energies growing of the
microorganisms (20.8 kJ/mol)), ethanol formation (35.8 kJ /mol), cell death (19.1
kJ/mol) are recorded as higher one from the previous data in the literature
(Phisalaphong et al. 2006) for the cells grown on mesophillic temperature and have
lower values for the same at the higher temperatures resistant microorganisms (Aiba
et al. 1973; Beadle et al. 1999; Geijnen 1999; Phisalaphong et al. 2006). Ea is known
as the important indictor for metabolic systems (Kelly et al. 2004). Most probably
mutagenesis enhanced the formation of these stabilizing proteins during product
formation, and this was done by the high temperature proteins of the metabolic
pathway within cells when growing at elevated temperatures as previously discussed
by Borges and Ramos (2005). For observing the protein profile of extra proteins
conferred by mutagenesis, intracellular extract of both native and mutant derivative
following growth on 15% total sugars in fermenter were run on 10 % SDS-PAGE
(Fig. 4.13). It was observed that not only invertaseband intensity was intensified in
the case of mutant, there were additional bands of 170, 78, 37 kDa and several bands
of molecular mass less than 32.5 kDa. Such proteins are secreted by thermotolerant
organisms as reported earlier by Borges and Ramos (2005) inductively as well as
constitutively.
92
Inv
175
83
62
47.5
32.5
Fig.4.13:
Intracellular
protein
expression
profile
of
derepressed
and
thermotolerant mutant M-9 on 15% TS with 3% corn steep liquor (lanes 1-2),
and native culture on this medium (lanes 3-4). M= protein marker and invert =
standard invertase from Sigma-Aldrich
Rajoka et al. (2005) also observed formation of additional proteins by the
thermotolerant mutant-derivative of S. cerevisiae during growth at higher
temperature. Many other researchers are of the observations that the microorganisms,
which are under the stress of elevated temperature can produce other products too
such as: glycerol and trehalose (Hounsa 1998; Aldiguier et al. 2004), though glycerol
production by the mutant cells was quite low but measurable (0.1 g/l h) (results not
presented).
93
4.10.1 Effect of temperature on ethanol production from molasses.

The data (Figures 4.14a to 4.14c) and (Tables 4.17 and 4.178) illustrate the time
course study of ethanol microorganism growth and sugar consumption by both the
organisms of wild and mutated one of S. cerevisiae at various temperatures. At 37 oC,
maximum cell mass was produced, along with maximum substrate consumption and
ethanol production (75 g/l) in response to the optimized inoculum size (10 % v/v).
When the temperature increased (42 -47 oC) the ethanol production decreased and a
minor decrease was recorded in the sugar consumption (145 to 140 g/l). However, 0.5
to 1.0 % sugar remained in the fermented broths when the experimental work was
ended for the process of fermentation at the end of fermentation. Fermentation results
at 45 oC and 47 oC are almost same with respect to sugar consumption and ethanol
production and the values have declined as compared to 43 oC in 28 h fermentation
time in case of mutant derivative .When the results of these experiments matched to
work of researchers of the time it was found the strain, presented by Banat et al.
(2002) and Rajoka et al. (2005). Another heat resistant microorganism developed by
another researcher (DAmore et al. 1989) and gave a reasonable amount of 6.8% that
of alcohol when fermented at temperature of 40 oC from 15 % total sugars utilized for
this experiment.
In another report of Joshi and Ashok (1999),K. marxianus gave 6.4-6.8 % alcohol for
the period of 42 h for the same temperature as in previous one. Additional beneficial
characteristics for the mutant strain counted as alcohol enhancement in productivity
and sugar consumption is also recorded as highest one at the elevated temperatures
(37-47 oC) when matched to wild organism for the same temperatures (Table 4.17 and
4.18). Because of both higher values of alcohol productivity and the sugar
94
consumption at 43 oC. These conditions are optimized for the elevated temperatures
(43-47 oC) for the batch fermenters of 23 liters. The results at higher temperatures
(Figure 4.17 and 4.18) are some what better than the values as mentioned in the
literature (Banat et al. (1998) and Rajoka et al. (2005) for the thermotolerant S.
cerevisiae in 28 h.
In thermophilic reactors, by increasing the temperature the
productivity decreases as shown by Banat et al. (1995, 1998, 2002) and Rajoka et al.
(2005). The results showed that a theoretical yield of 98.03 % of ethanol was achieved
under these investigations and this value is higher from the values already presented
(Rajoka et al. 2005) in the earlier publications. These results confirmed that this strain
has the potential to be used on commercial scale under controlled conditions of
process variables.
95
Table4.17: Effect of different representative temperatures on ethanol and cell

mass formation during time-dependent sugar consumption of sugars from
molasses medium by both wild and mutant strains of S. cerevisiae.
Time
37 oC
(h)
12
16
20
24
28
40 oC
43 oC
45 oC
47 oC
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
150
150
150
150
150
150
150
150
150
150
0.2
127
0.2
127
0.2
127
0.2
127
0.2
127
0.84
132
0.84
132
0.84
132
0.84
132
0.84
132
1.6
111
11
1.6
111
1.6
111
1.6
111
1.6
111
3.8
102
26
3.8
102
13
3.8
102
10
3.8
102
12
3.8
102
90
17
90
10
90
90
90
3.9
62
40
3.9
62
21
3.9
62
34
3.9
62
15
3.9
62
15
3.7
35
28
3.7
35
11
3.7
35
18
3.7
35
3.7
35
4.7
21
57
4.7
21
34
4.7
21
31
4.7
21
25
4.7
21
17
4.8
17
39
4.8
17
21
4.8
17
24
4.8
17
4.8
17
5.9
10
68
5.9
10
45
5.9
10
36
5.9
10
21
5.9
10
19
7.0
11
41
7.0
11
32
7.0
11
31
7.0
11
11
7.0
11
7.4
74
7.4
75
7.4
71
7.4
23
7.4
20
7.0
39
7.0
32
7.0
30
7.0
11
7.0
7.4
74
7.4
73
7.4
71
7.4
65
7.4
58
Table4.18: Temperature-dependent kinetic parameters for ethanol and cell mass

formation during substrate consumption (15% total sugars in molasses, pH=5.5)
by the mutant strain of S. cerevisiae
Temperature
o
C
37
40
43
45
47
Qx
Qs
Qp
Y X/S
YP/X
YP/S
qp
qs
0.35
0.33
0.26
0.21
0.18
0.33
0.32
0.32
0.29
0.26
5.2
5.25
5.17
5.00
4.7
2.64a
2.60b
2.53c
2.32d
2.07e
0.063
0.061
0.062
0.057
0.055
7.88
8.02
7.89
8.02
7.84
0.5
0.49
0.48
0.46
0.43
0.76
0.65
2.05
1.68
1.41
5.5
5.4
4.19
3.69
3.27
F value = 3162 (p =0.000) for Qp LSD = 0.02
96
Fig. 4.14(a): Effect of temperature on the production of ethanol

from 15 % sugars under optimized conditions
97
Fig.4.14 (b): Effect of temperature on cell mass formation from 15% sugars
under optimized conditions
98
Fig.4.14: Effect of temperature on substrate consumption from 15% sugars

The growth of S. cerevisiae and its mutated strain were highly affected by
temperatures during Ffase production. Enzymatic activation at 45C found less and
cell formation temperature was recoded as 40 C. Production of the enzymes from the
mutated strains found their maximum values as compared to the native strains at all
temperatures.
4.11 THERMODYNAMICS OF ETHANOL PRODUCTION PROCESS
4.11.1 Thermodynamics of ethanol production from hydrol and molasses
Under controlled environmental conditions, these parameters are vital for the
microorganism growth and productivity (Von Stocker et al. 2006).The changing the
state stable organism has been suggested that the possible mechanism, which may
explain when it needed in the increase of production rate when the temperature goes
high (Benkovic and Hammes-Schiffer 2003). Temperature has been suggested to
99
carry substrate and bind it for the changing nature of the microorganism. This
complex then changes the resisting factors for the production of ethanol and enzymes
under such conditions (Garcia-Viloca et al, 2004). Entropic guidance, and increase in
the generalized transmission coefficient are also considered to significantly contribute
in enhancement of reaction rates in enzyme catalyzed reactions in the product
formation metabolic network (Garcia-Viloca et al, 2004) and may give a clue to
thermostability.Alberty (1998) has worked for ht stability and thermal stability of the
microorganisms and found a better conditions in the steady state.Arrhenius model was
utilized for finding out the values of these energies and hidden thermal process
regulatory mechanism (Aiba et al. 1973; Converti and Dominguez 2001). The best
specific output addressed from the crossing values in the interference of the two
straight lines (Fig.4.15a), representative ever-increasing and declining specific
production magnitudes about the best. Curves were used to calculate the values of
enthalpy of activation/inactivation equilibrium, while the intercept values were used
to calculate the entropy demands of activation/inactivation equilibria.Enlarge values
of 40 oC have given highest production after mutations. From the Fig. 4.15. (Hydrol &
molasses) enthalpy was calculated for activation energy of alcohol productivity
network and that was presented in a graph.
100
Fig.14.5 (a): Enthalpy and entropy requirements for alcohol production from
hydrol in the shown temperature ranges. An average is shown in the data, =
parental and = mutant culture as per Arrhenius equation.
101
Fig. 4.15(b): Enthalpy and entropy requirements for alcohol production from
hydrol in the shown temperature ranges. An average is shown in the data, =
parental and = mutant culture as per Arrhenius equation.
The factors presented (Table 4.19) show that (H*
for the mutated strain is
37.4KJ/mol from hydrol was lower than that on molasses (187 kJ/mol) and for
phytase production ((H*70-80 KJ/mol) as presented earlier (Converti and Dominguez
2001)and it was compared with other bioprocess .For example growing of the strain
(34-74KJ/mol) as reported by previous workers (Aiba et al, 1973 and Converti and
Dominguez 2001).
102
Table 4.19: Growth on hydrol and molasses substrates according of Arrhenius.

H* (kJ/mol)
Organism
S*(J/mol.K)
Alcohol
Thermal
Alcohol
Thermal
Production
Inactivity
Production Inactivity
Hydrol
37.4
59.0
-122.6
-430.2
Molasses
187.0
n.d.
-534.2
n.d.
Hydrol
36.6
33.3
-126.6
-358.7
Molasses
69.8
n.d.
-841.8
n.d.
Parent
Mutant
The activation entropy of mutant for ethanol formation (-122.6 J/mol/K) is extremely
lesser value and compared to xylitol reaction by thermotolerant yeast (Converti and
Dominguez 2001) but lower than several other fermentation processes (von Stockar et
al. 2006). All these informations proposed this mutation gave valued enhancement to
the microorganisms.
When organism was grown at a temperature of 37-40C.If any inactivity occurs. The
enzymes may reach there for the best achievements. I thermal inactivity which is a
result of concomitant increase in the enthalpy of activation (Chen and Stites 2000;
Vieille and Zeikus 1996). The defolding of the enzyme structures of metabolic
network is accompanied by an increase in the disorder, aggregation of proteins or
increase in entropy of activation (Chen and Stites 2000). .Value of heat parameters of
inactivation found with the help of Fig. 4.15 presented a value which was less than the
production. The phenomenon characterized through an inactivation enthalpy valued
as 36.6 kJ/mol in case of mutationally derived strain during the production of alcohols
metabolically. This behavior according to Aiba et al. (1973) is demonstrated by
103
thermotolerant organisms. Defolding of proteins of metabolic network) does not

increase faster with increase .This data for the energy is very week for the system(
Converti and Dominguez 2001).
Above statements signify that rate of deactivation of the glycolytic pathway not
increased faster as the light with the updating in temperature. Showing the metabolic
networks conformational stability. It also makes the values lower obtained with the
microbial processes (160-235 kJ/mol) (Von Stockar et al. 2006). Mutated
microorganism has very low values pf the thermal inactivation by the mutant culture
is lesser value (-444.4 J/K.mol) was lower than that needed by native strain which has
had negative symbol. In both native and mutant strains, entropy value of thermal
inactivation was found to be lower than that of alpha amylase (Declerck et al. 2003).
This also indicated that the mutant organism may have acquired the ability to exert a
sort of safety for the thermal in activation
perhaps by heat shock proteins
,chaperones, in the cells .(Chen and Stites 2000),therefore the mutational changes
have made the system very strong during the alcohol production from hydrol..That
was very different with the observations of the previous literature (Allen et al.
1989).It was also concluded by Ricon et al (2001) that the resistance to DG will
change the shifting system of the enzymes an d are very stable in the medium for the
fermentation.
4.11.2 Thermodynamic parameters of extra-cellular Ffase production.
Demand for Ea increase, low inactivation heat energy requirement of cultural
endogenous metabolism and lowe level requirement for the enthalpy and entropy are
very important for thermo-stability when the productivity of enzymes stable(Siddiqui
et al. 1997).When Ea = 36 kJ.mol- it was obvious that the work was many fold
104
stable(1..3 fold) and when that of wild culture is Ea = 46 kJ.mol-1 on molasses. That
was less than the requirement of the cultures. (Aiba et al. 1973). Thermostability of
the production system can also been confirmed by Fig. 4.16
Fig.4.16: Arrhenius relationship to calculate enthalpy and entropy of activation

for invertase production and inactivation pathway.
The value of the enthalpy of extracellular Ffase formation by mutant (H *=53.5 k J/
mol) is less than the wild organism (67.1 k J mol-1).Cell growth, Enzyme production
and the product (70-80 k J/mol) (Converti and Dominguez 2001) (Aiba et al. 1973)
and products released in fermentation medium (Converti and Dominguez 2001).
Phenomena responsible for inactivate Ffase formed by mutant cells have the
properties of this energy of activation and that is very low at its value for the
production by wild cells (70.0 k J/ mol) (Converti and DelBorghi 1996).
105
Table 4.20: Enthalpy and entropy values of extracellular Ffase production and
inactivation pathway following growth on molasses at different temperatures.
Parent
67.1
70.0
S*(J/mol.K)
Product
Inactivity
Formation
fFase
12.6
-365.4
Mutant
53.5
52.4
-26.4
Organism
H* (kJ/mol)
Ffase
Thermal
formation
inactivation
Ffase production though the mutated cells
-428.4
(-0.0264 k J/mol K) is very low and
ethanol and xylitol needed. (Converti and Dominguez 2001). Entropy is required for
the purpose and that is -0.428.4 k J/mol K) is also very small and is visible. It is
observed that this could be a lower value as ethanol is produced by heat resistant cells.
(Converti and Dominguez 2001; Rajoka et al. 2005). This suggests a sort of an
effective safety on mutated cells. As shown in Fig.4.17 that mutation conferred
genetic ability on the wild organism of producing additional proteins, which may be
heat shock proteins. Such proteins have been reported to keep the proteins in folded
form when exposed to higher temperature (Borges and Ramos 2005).
106
4.12. EFFECTIVENESS OF pH FOR ALCOHOL & FFASE PRODUCTION

Filamentous fungi and yeasts have competent system of pH and ion homeostasis.
These mechanisms allow them to grow in such and environmental conditions vary
(Perez-Gonazalez et al. 1998). Throughout this acceptance, they have capability for
PacC .pH plays a vital role for the gene expression when it is acidic (Perez-Gonazalez
et al. 1998).The ranges for the pH are 3.4 to 8.00 where as the maximum value for the
ethanol and enzymatic production. for controlled and uncontrolled conditions.(and
controlled at 5.5) is regarded as optimal for ethanol and Ffase production in 23 liter
fermenter.The optimum pH of ethanol and Ffase productivity was comparable with
that reported earlier (Euzenat et al. 1997; Hayashi et al. 1992; Rincon et al. 2001;
Rajoka et al. 2005).
Fig.4.17: Effect of controlled pH on Ffase formation by parental () and mutant

() culture and ethanol production under optimized conditions of aeration and
agitation.
107
Each value is a mean of two independent readings. Error bars show standard error
between them.pH was optimized as 5.5 and enzymatic production was pH 6.5.and a
separates study was revealed suitable for the production case.
4.13. ETHANOL AND FfASE IN 150 LITER FERMENTER FOR THE

PRODUCTIVITY
Scaling up of the experiment is crucial for any study related to fermentation that is
aimed at the hyper production of a particular product. Production of ethanol by S.
cerevisiae is an anaerobic process but requires sufficient mass of dissolved oxygen in
the medium for ethanol production. Under the aeration rate of 1.0 vvm (optimized),
for 8 h, followed by 0.25 vvm for other 20 h fermentation was optimum in the 15-litre
fermenter cultures. Ethanol production was associated with yeast growth for 8 h and
then was not associated with growth for the next 20 h.
Fig.4.18: Representative time course production of ethanol by native () and

mutant (), cell mass by native () and mutant () with consumption of sugars
by native () and mutant ()in 150 liter fermenter using hydrol as a carbon
source at 40 C.
108
Hydrol was treated with Dextrozyme (1 Unit/g substrate) for 1 h at 60 C, followed by

simultaneous saccharification and fermentation at 40 C. Qp was 3.5 and 2.2 g/l h by
the mutant and native organism respectively with cell mass rates of 0.34 and 0.24
g/l.h with sugar uptake rate of 4.6 and 3.2 g/l.h respectively. Potential S. cerevisiae
during growth in optimized molasses media in 150 liter fermenter studies indicated
that molasses supplemented with ammonium sulfate supported 1.55-fold higher
volumetric productivity than that by unoptimized medium and that in 150 liter
fermenter aeration and stirring enhanced enzyme titre by 1.33-fold over optimized
media in 23 liter fermenter.
Table 4.21: Dependence of ethanol production on culturing condition namely 23
liter,150 liter fermenter and shake flask (s. flask) cultures: Kinetic parameters
for substrate consumption (molasses) and ethanol formation by S. cerevisiae
mutant derivative.
Parameters
23 liter
150 liter
S.flask
F-value
p- value at
p0.05
Substrate consumption parameters

m(h-1 )
0.34b
0.35a
0.23c
441
0.000
Q S (g/L h)
4.5b
6.25a
3.8c
204.8
0.0001
YX/S (g/g)
0.06b
0.066a
0.045c
263.3
0.0000
qS
5.7a
5.5a
5.6a
0.12
(g/g h)
0 .459
Product formation parameters

QP* (g/l h)
3.38b
4.5a
2.4 c
331.24
0.0001
Y P/S (g/g)
0.49b
0.50a
0.45c
63.0
0.000
Y P/X (g/g)
7.9b
8.30a
8.10c
183.0
0.0001
qP* (g/g h)
2.7b
3.0a
1.7c
139.0
0.0002
Lane 1: Each columns 1, 2, and 3 Duncan multiple range test applying ANNOVAII in
MstatC software (n=2).
109
Furthermore, the substrate uptake rate was improved 1.39 and 1.64-fold over that in
23 liter and shake flask cultures respectively. The influence of treatments on all
fermentation attributes of ethanol production was highly significant except for
(Aldiguier et al. 2004;Mizuno et al. 2006; Phisalaphong et al. 2006;Rashad
2003;Sridhar et al. 2000), K. marxianus and its mutant (Banat et al. 1998; Limtong et
al. 2007). Ethanol volumetric productivity, ethanol yield and theoretical yield on
optimized medium in 150 liter fermenter were 4.5 g/l.h, 0.50 and 98 % using 150 g
TS/liter in molasses at 40 C. Previously, it was documented that S. cerevisiae FB at
40-43 C supported ethanol yield of 0.40 g/g from 170 g sugars/liter and S. cerevisiae
F29 yielded only 0.28 g/g product yield under same fermentation conditions (Banat et
al. 1998). Banat et al (1998) reported three strains of S. cerevisiae Fill, S. cerevisiae
WR12 and S. cerevisiae SIIC which produced 0.45, 0.47 and 0.45 g/ethanol/g sugar at
40-43 C following growth on 175, 171 and 159 g TS/liter respectively. Abdel-fattah
et al. (2000) performed extensive studies with S. cerevisiae Fill at 30-45 C using 160
g TS/liter and got 0.48 g ethanol/g TS and was remarkably good performance of the
culture at industrial scale. Their studies on S. cerevisiae SIIC gave very low ethanol
yield (0.15 g/g) at industrial scale. However Kluyveromyces marxianus under similar
conditions supported 0.41 g ethanol/g TS at 40-45 C. Previously Rajoka et al. (2005)
isolated a thermotolerant mutant of S. cerevisiae, which supported an ethanol yield of
0.49 g/g at 37 -43 C in 23 liter fermenter. Thus ethanol yield of 0.49g/g by mutant S.
cerevisiae M-9 from 150-200 g TS/liter at 40-47 C is quite appreciable. Ethanol
production regulatory process was comparable to that exhibited by a Bacillus sp.
(Gupta et al. 2004). The production formation a main mot Enhancement in ethanol
110
and Ffase production by the mutant was a consequence of alteration in genes related
to all activities, namely hexokinase, or DOG-6Phosphatase as reported in DGr
mutants (Rincon et al. 2001).
Invertase production on molasses inexpensively for food industry in Pakistan and
ethanol as solvent for several industries. Both extra-cellular and intracellular fructofuranosidase discovered their hyper-productivity from molasses under which wild
organism is not appropriate for use.
Modification in cell wall, making proteins or cell membrane is a natural phenomenon
for the resistance to the unwanted materials through wall and membrane. (Rincon et
al. 2001). That leads to increase the production of cells. Mutated cell has been utilized
more effectively as compare to the native or wild one. Utilize sucrose, fructose and
glucose or molasses and -fructo-furanosidase. The results presented in this work are
better as compare to the researchers (Allen and Roche 1989).
111
CHAPTER 5
CONCLUSIONS & RECOMMENDATIONS
5.1 CONCLUSIONS
A commercial yeast strain S. cerevisiae was mutated by Gamma radiation to impart
ability to hyper-produce ethanol and simultaneously increase its thermostability. Two
approaches were employed for obtaining the desired phenotypes of the wild or native
organism. Out of mutagenizing -ray dose, 1 kGy was selected as the best exposure
dose for effective mutant selection and that first expression using larger cell volume
of exposed cell population was more effective and was finally employed for getting
the desired genotypes and mutant derivative finally selected was designated as M9.This improved mutant was selected in the laboratory and semi commercial scales.
The mutant strain proved more productive with respective to ethanol i.e., 7.5 % (w/v),
95.4 % (w/w) of the theoretical yield and 9.4 (w/v) cell mass and consumed almost all
sugars i.e; 98.6% (w/v) at 43 oC. The mutant strain along with the native culture was
utilized for the optimization of other parameters at 23 liter fermenter (15 liter working
volume). At the large scale fermentation unit and investigating various fermentation
parameters, such as carbon sources and their concentrations, nitrogen sources, air flow
rate, agitation intensity, additives, and temperature on the production of ethanol and
cell mass with time course dependent substrate uptake. Glucose and fructose released
by the wild cells caused feedback inhibition which was overcome by DG-mutation.
Mutant cells act as an inducer, which could interface CreA-DNA as presented by
other workers (Bohm and Boos 2004; deVries et al. 1999). Standard substrate
112
concentration was done and 15% total sugar is observed as the best sugar
concentration in molasses to produce ethanol (74 g/l). It was recorded that lower the
concentration, lower the fermentation results (34% ethanol). Presence of more sugars
is ineffective and the system needs more time for fermentation and for ethanol
fermentation. Molasses and hydrol were used as C-sources to see the effects of the
substrate on ethanol fermentation. Molasses gave 74 g/l and hydrol gave 58 g/l of
ethanol. For the substrate consumption also, molasses proved better. This comparison
is first ever reported in the ethanolic fermentation at pilot scale. Though 15% sugars
in molasses was selected as the best concentration for optimization studies, the mutant
was insignificantly different in supporting ethanol from 15, 17.5 and 20 % sugars in
molasses and catabolite repression. The wild organism was significantly repressed for
ethanol production from 17.5 and 20% sugars in molasses. Thus CreA proteins were
effective in controlling gene for the native organism. The mutated strain was derived
was devoid of such catabolite repression.
Diammonium sulfate was recorded as the best nitrogenous source and produced 75 g/l
ethanol, 9.4 g/l cell mass after utilizing 148 g substrate/l. Ammonium salts cause
nitrogen source catabolite repression but in this case both native and mutant strains
were ammonium ion catabolite repression resistant. The effect of air flow rate was
also investigated and the ethanol and cell mass were recorded as 70 g/l and 7.4 g/l cell
respectively consuming upto 89 % at 0.1 volumes at optimum
variables. The
addition of NaF and Tween 80 as additives had no major effects on the production of
ethanol; however, a slight enhancement in the production of ethanol was achieved
when Tween 80 was used.. An increase in the reaction time for up to 72 h may utilize
more sugars and ultimately produce more ethanol.
113
This thermotolerant yeast strain was utilized at elevated temperatures for testing its
thermo-tolerance. At 37
C, maximum cell mass produced with concomitant
maximum ethanol production (75 g/l). Maximum substrate was consumed in response
to the optimized inoculum size (10 % v/v).At the ranges of the temperatures i.e.42-47
o
C) the ethanol production decreased in the native organism and a minor decrease was
recorded in the sugar consumption too (145 to 140 g/l). However 0.5 to 1.0 % sugar
remained in the fermentation broths in these cases. The results of the fermentation at
45 oC and 47 oC (by the mutant) are almost the same with respect to sugar
consumption; however the ethanol production values have declined as compared to
those at 43 oC in 28 h fermentation time.
The rate for mixing at 300 rpm in the reactor was optimal. The outcomes of common
products of the experiment at a variety of temperatures checked with the Arrhenius
equation exposed that alteration in the yeast specie has minimized the degree of
enthalpy and entropy requirements for the inactivation equilibrium during product
formation, suggesting that mutation made the metabolic network of the organism
thermally more stable. The highest magnitudes of volumetric productivity and other
product attributes of ethanol and invertase formation occurring on optimized medium
in fermenter are greater than data a given by other researchers. Activation energy,
enthalpy and entropy of ethanol formation at 50 C, and magnitudes of Gibbs energy
and the melting point indicated that the metabolic pathway of ethanol formation
(dependent partially on invertases and is highly thermostable.
The mutant produced through radio activity of gamma rays are proved stable rayinduced mutation has given a stable and viable mutant for hyper-production of Ffase
114
and alcohol; the productivity is more than 2 fold improved as compare to the wild
strain.
5.2
RECOMMENDATIONS
On molasses-based medium for extracellular and intracellular Ffase production and

1.3-fold for alcohol production using commercial alcohol production conditions. This
improvement may be because of gene copying number or gene improvement or
improvement in gene expression or both or improvement in transport system of the
cells. This hyper- secretion is of has a great importance and more study is needed.
This improved strain of S. cerevisiae produces hyper of Ffase and alcohol .This gene
could be useful for the further treatment and study for improvement .It may be
summarized that this strain would serve the purpose of commercial scale production
of ethanol using a cheep source of ethanol production from sugar cane molasses in our
countryGenome transfer is an influential approach for imperative modification of
microbial strains for desirable industrial phenotypes. Further improvement in heat
resistance and ethanol acceptance of commercial scale yeast may be brought about by
this technique (Shi et al. 2009). In this technique, starting population can be generated
by ultraviolet treatment of protoplasts and then subjecting them for the recursive
blending. Screened colonies may be utilized for the growth at a temperature of 35, to
55 C with the range of 5 degrees in each. This way, thermo-tolerance and ethanol
tolerance may be enhanced in the newly developed recombinants, which may grow
and utilize 20% (w/v) glucose at 45-48 C, to produce 9.95% (w/v) ethanol, and
tolerating 25% (v/v) ethanol stress (Shi et al. 2009).
115
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ENHANCED PRODUCTION OF ETHANOL FROM

SUGAR CANE MOLASSES THROUGH THERMOTOLERANT

SYED FARMAN ALI SHAH

In fulfillment of the requirement for the degree of

Department of Chemical Engineering

1.2 Ethanol and its scope

1.3 Ethanol production

1.4 Raw materials for ethanol production

1.5 Fermentation by yeast, S. cerevisiae

1.6 Aeration in fermentation

1.7 Oxygen transfer

1.8 Determination of KLa value

1.9 Why thermotolerant yeast is used?

1.10 General modification of strain

1.11 Objectives of present research

CHAPTER 2 LITERATURE REVIEW

2.1 Ethanol and its by-products

2.2 Yeast and invertases

2.3 Other parameters

2.3.1 Substrate concentration

2.3.2 Nitrogen and carbon sources

2.3.3 Airflow rate

2.3.5. Thermodynamics of ethanol and Ffase

CHAPTER 3 MATERIALS AND METHODS

3.1 Research centers

3.2 Sample centers

3.3 Microbial strain

3.4 Maintenance of culture

3.5 Growth medium composition

3.6 Preparation of plates

3.7 Preparation of slants

3.8 Preparation of the culture of native S. cerevisiae

Preparation of yeast growth medium

3.9 Effect of gamma rays irradiation on viability of

3.11 Purification of mutants of S. cerevisiae

Slants of purified culture

3.12 Propagation of yeast

First stage propagation

Second stage propagation

3.13 Effect of carbon and nitrogen sources on ethanol

both wild and mutant culture in 23 liter fermenter

3.16 Analytical methods

Preparation of standard curve for

Ethanol estimation through

Determination of units for

(Dinitrosalicylic Acid) solution

Standard curve of glucose

3.17 Chemical composition of hydrol (starch molasses).

3.18 Determination of growth kinetic parameters

3.19 Effect of temperature

3.20 Determination of thermodynamic parameters

3.20.1 Thermodynamics of cell mass and

CHAPTER 4 RESULTS AND DISCUSSION

4.1 Mutagenesis of S. cerevisiae using -rays

4.2 Substrate regulation of invertase and ethanol

4.4 Substrate concentration dependent formation of

4.6 Regulation of ethanol production by nitrogen

4.8 Effect of Agitation

4.9 Effect of additives

4.10 Production from hydrol

Effect of temperature on ethanol

4.11 Thermodynamics of ethanol production process

Thermodynamics of ethanol production